EXPERIMENTAL  PHARMACOLOGY 


GREENE 


EXPERIMENTAL  PHARMACOLOGY 

A  LABORATORY  GUIDE 


FOE  THE  STUDY  OF 


THE  PHYSIOLOGICAL  ACTION  OF  DEUGS 


BY 

CHARLES  WILSON  GREENE,  PH.  D. 

PROFESSOR  OF  PHYSIOLOGY  AND   PHARMACOLOGY,   UNIVERSITY  OF  MISSOURI. 


THIRD  EDITION,  REVISED 
WITH   37  NEW  ILLUSTRATIONS 


PHILADELPHIA 

P.  BLAKISTON'S  SON  &  CO. 

1012   WALNUT  STEEET 
1909 


& 


COPYRIGHT,  1909,  BY  P.  BLAKISTON'S  SON  &  Co. 


Printed  by 

The  Maple  Press 

York,  Pa. 


PREFACE. 


Instruction  in  Pharmacology  should  be  based  on  a  rigid  course  of 
required  laboratory  experiments.  The  student  in  the  subject  must  be  given 
every  opportunity  to  observe  for  himself  the  changes  produced  by  a  drug  in 
the  activities  of  a  tissue,  of  an  organ,  and  of  the  entire  organism.  It  is  only 
on  such  intimate  personal  experience  with  the  facts  that  one  can  reach  a 
rational  understanding  of  the  principles  of  Pharmacology. 

The  directions  presented  here  have  been  formulated  during  the  growth 
of  the  course  as  presented  in  the  University  of  Missouri.  Under  each  drug 
presented  there  is  given  a  list  of  experiments  chosen  with  due  consideration 
to  the  facility  with  which  they  may  be  performed  by  students.  This  list 
is  followed  by  detailed  yet  brief  directions  for  the  execution  of  the  experi- 
ments. An  occasional  type  illustration  is  given  to  guide  the  student  in  his 
efforts.  It  is  assumed  that  the  individual  student  will  have  time  for  only  a 
selected  number  of  experiments  on  each  drug,  and  the  plan  of  the  Guide  is 
so  arranged  as  to  support  the  instructor  in  the  assignment  and  execution  of 
a  number  of  diversified  experiments  by  the  average  laboratory  class. 

I  am  indebted  to  the  Department  Teaching  Staff,  especially  to  Professor 
W.  Koch,  now  of  the  University  of  Chicago,  to  Dr.  W.  H.  Schultz,  now  of 
the  U.  S.  Public  Health  and  Marine  Hospital  Service,  and  to  Professor 
R.  B.  Gibson  for  numerous  suggestions  and  much  assistance. 

I  am  under  special  obligation  to  Mr.  G.  T.  Kline  for  the  drawings  of 
apparatus,  to  Professor  Gibson  and  to  my  students  of  the  past  five  years 
for  the  majority  of  the  new  illustrations  presented  in  this  edition  of  the 
Guide.  C.  W.  G. 

UNIVERSITY  OF  MISSOURI,  April,  1909. 


no-t 


CONTENTS. 


THE  ACTION  OF  DRUGS. 

PAGE 

Alcohol i 

Ether 6 

Chloroform    .    .    . n 

Chloral  hydrate 14 

The  opium  series 15 

Caffeine 20 

Strychnine      24 

Cocaine 29 

Quinine 32 

Atropine,  scopolamine 33 

Nicotine 37 

Curare 40 

Pilocarpine 42 

Physostigmine 44 

Aconite 48 

Veratrine 50 

Digitalis 52 

Ergot 55 

Suprarenal  gland 56 

Nitroglycerine  and  the  nitrites 59 

Carbolic  acid 61 

Potassium  salts 61 

Calcium    salts 62 

Barium  salts 63 

OPERATIONS,  APPARATUS  AND  SPECIAL  METHODS. 

Physiological  solutions 64 

Anesthesia  for  dogs,  cats,  rabbits,  and  guinea-pigs 65 

The  preparation  of  the  ventricular  muscle 66 

To  test  the  action  of  drugs  on  the  frog's  heart 68 

vii 


Vlll  CONTENTS. 

PAGE 

Irrigating  and  perfusing  flasks  .  .  . 69 

To  test  the  action  of  drugs  on  the  blood-pressure,  respiration,  etc.,  of 

a  mammal - 70 

Method  of  testing  the  action  of  drugs  on  the  reflexes  of  a  frog 71 

Method  of  giving  and  testing  the  action  of  a  drug  on  the  frog's  gas- 

trocnemius  muscle 72 

Transfusion  buret  for  mammals 72 

Apparatus  for  the  study  of  the  isolated  mammalian  heart  .....  73 

List  of  stock  solutions 75 

Report  form  for  experiments  on  frogs 76 


LIST  OF  ILLUSTRATIONS. 


. 

1.  Alcohol  perfused  through  the  frog's  heart 2 

2.  Ether  on  the  isolated  muscle  of  the  terrapin's  ventricle  .....  7 

3.  Strong  ether  solutions  on  the  isolated  ventricular  muscle 8 

4.  Ether  on  tone  waves  of  auricular  and  sinus  muscle 9 

5.  Ether  on  the  irritability  of  the  sciatic  nerve 10 

6.  Chloroform  on  the  rhythm  of  heart  muscle 12 

7.  Chloroform  perfusion  of  the  frog's  heart 13 

8.  Chloroform  on  the  irritability  of  nerve 13 

9.  Morphine  on  the  rhythm  of  terrapin's  ventricle 16 

10.  Morphine  on  the  isolated    heart  of  the  cat .    .  17 

11.  Morphine  on  the  isolated  heart  of  the  cat 17 

12.  Caffeine  on  the  ventricular  muscle 21 

13.  Caffeine  on  the  amount  of  muscular  work 22 

14.  Strychnine  on  the 'ventricular  muscle 25 

15.  Strychnine  on  the  muscular  work 26 

1 6.  Strychnine  on  the  isolated  mammalian  heart 27 

17.  Cocaine  effects  on  the  frog's  heart 29 

1 8.  Cocaine  on  the  muscular  work 30 

19.  Quinine  on  the  frog's  heart 32 

20.  Atropine  on  the  heart  muscle 33 

21.  Atropine  on  the  isolated  cat's  heart  . 34 

22.  Nicotine  on  the  ventricular  muscle 38 

23.  Nicotine  on  the  isolated  cat's  heart 39 

24.  Pilocarpine  on  the  blood-pressure  and  on  the  respiration  of  the  dog  42 

25.  Physostigmine  on  the  isolated  cat's  heart 45 

26.  Physostigmine   followed  by  atropine  on  the  mammalian  blood- 

pressure  and  respiration 46 

27.  Aconite  on  the  isolated  cat's  heart 49 

28.  Veratrine  on  the  isolated  cat's  heart 51 

29.  Digitalis  on  the  heart  muscle 53 

30.  Digitalis  on  the  isolated  cat's  heart .....  54 

ix 


X  LIST   OF    ILLUSTRATIONS. 

FIG.  PAGE 

31.  Adrenalin  hydrochloride  on  the  isolated  cat's  heart    ......  57 

32.  Amyl  nitrite  on  the  human  pulse 60 

33.  Calcium  chloride  on  the  terrapin's  heart  muscle 62 

34.  The  terrapin's  heart,  how  to  cut  an  apex  strip 67 

35.  Apparatus  for  the  study  of  heart  muscular  strips 67 

36.  Apparatus  for  the  study  of  drugs  on  the  frog's  heart 68 

37.  Apparatus  for  the  study  of  the  action  of  drugs  on  the  mamma- 

lian heart 74 


EXPERIMENTAL 
PHARMACOLOGY 


THE  ACTION  OF  DRUGS. 

ALCOHOL. 

List  of  Experiments  Showing  the  Effects  of  Alcohol.  PAGE. 

1.  On  the  frog i 

2.  On  ventricular  muscle      ;.-r.:-t •,.;..: i 

3.  On  the  frig's  heart  .    .':•.:   V-£>Viv  ;•/>••-  .....  2 

4.  On  the  isqlated  mammalian  heart,  ..;    .  '..  .    ......  3 

5.  On  the  work  of  the  frog's  gastronemius  muscle 3 

6.  On  voluntary  work  of  human  muscle.     Demonstration .    .  4 

7.  On  the  circulatory  and  respiratory  systems  of  the  mammal .  4 

8.  On  the  reaction  time  of  the  reflex  frog     .    . 5 

1.  Alcohol  on  the  frog.     Inject  into  the  dorsal  lymph  sacs  of  two 
frogs  doses  of  0.3  c.c.  (5  minims)  and  0.6  c.c.,  respectively,  of  95  percent 
alcohol.1     Strong  alcohol  is  quickly  absorbed  from  the  lymph  sac.     The 
larger  dose  is  sufficient  to  produce  temporary  complete  loss  of  the  reflexes, 
together  with   the  loss  of  all  respiratory  movements.     A  dose  of   I   c.c. 
is  toxic  for  a  4o-gram  frog.     Since  the  smaller  dose  is  equivalent  to  525  c.c. 
for  a  yc-kilo  man,  it  is  evident  that  the  frog  is  the  more  tolerant  of  alcohol.2 

2.  Alcohol  on  ventricular  muscle.     Mount  a  strip  of  the  ventricle 
or  a  terrapin  in  0.7  percent  sodium  chloride  (see  page  66  for  the  method), 
and  when  it  is  contracting  with  an  even  and  regular  rhythm  change  to  a 
solution  of  2  percent  alcohol  in  physiological  saline.     Return  the  strip  to 

JA11  doses  for  frogs  given  in  this  book  are  calculated  for  an  animal  weighing  40  grams. 
Each  animal  used  for  experiment  should  be  weighed  on  a  "  Harvard  "  platform  balance  and 
the  dose  given  calculated  in  proportion  to  the  weight  of  the  animal  used. 

2  A  report  blank  form  for  the  tabulation  of  observations  on  the  effect  of  drugs  on  frogs  is 
given  on  page  76. 


2  EXPERIMENTAL    PHARMACOLOGY. 

pure  saline  solution  after  two  to  five  minutes.  Record  the  contractions  on  a 
drum  moving  i  mm.  a  second.  Repeat  the  experiment,  using  successive 
strengths  of  alcohol  of  5  and  10  percent.  The  alcoholic  effect  will  be  dem- 
onstrated rather  better  on  a  ventricular  strip  that  is  contracting  regularly 
in  the  weaker  Ringer's  solution,  page  64,  but  the  alcohol  must  be  dissolved 
in  Ringer's  solution  of  the  same  composition. 

3.  Alcohol  on  the  frog's  heart.  Destroy  the  brain  and  spinal  cord 
of  a  frog,  expose  the  heart  by  cutting  away  the  ventral  wall  from  directly 
over  the  ventricle,  using  care  not  to  lose  blood.  Do  not  cut  the  bridge  formed 


* 


FIG.  i. — Action  of  2  percent  alcohol  on  the  frog's  heart  when  perfused  through 
the  ascending  vena  cava.  The  alcohol  was  dissolved  in  Ringer's  solution.  The 
record  was  taken  from  the  tip  of  the  ventricle  by  the  suspension  method.  Time  in 
seconds  and  minutes.  Perfusion  began  at  the  first  arrow  above  the  second's  record  and 
ended  at  the  second  arrow. 

by  the  sternum,  but  use  it  as  a  fixed  point  to  which  the  heart  may  be  anchored 
by  a  ligature  around  one  of  the  aortic  arches.  Take  a  direct  record  of  the 
movements  of  the  ventricle,  using  a  light  straw  lever  of  the  fulcrum-power- 
weight  order.  Give  1.5  c.c.  of  95  percent  alcohol  in  the  abdominal 
cavity.  Take  a  continuous  record  during  the  time  of  absorption.  This 
method  demonstrates  the  effects  on  the  volume,  and  on  the  type  of  systole 
and  diastole.  The  rate  is  only  slightly  changed. 

More  satisfactory  results  are  obtained  by  perfusing  the  heart  through  a 
canula  in  the  ascending  vena  cava.  Perfuse  the  alcohol  from  four-ounce 
supply  flasks  provided  with  constant  level  tubes.  Use  a  pressure,  of  from 
4  to  6  cm.  The  perfusion  strength  to  use  by  this  method  is  2  to  5  percent 
alcohol  made  up  in  Ringer's  solution.  Perfuse  the  heart  for  from  two  to 


ALCOHOL.  3 

four  minutes  at  a  time.  Record  the  contractions  of  the  ventricle  by  a  thread 
from  its  tip  to  the  vertical  arm  of  a  balanced  lever,  page  68.  In  this  experi- 
ment, as  in  all  frog's  heart  perfusion,  it  is  better  to  use  the  weaker  Ringer 
for  the  normal  solution.  The  Ringer's  solution  insures  a  uniform  heart 
rate  and  strength  for  long  intervals,  while  the  sodium  chloride  solution 
will  sustain  the  whole  heart  in  regular  and  strong  rhythm  for  only  a  few 
minutes. 

4.  Alcohol  on  the  isolated  mammalian  heart.     Use  a  rabbit  or  a 
cat  for  this  experiment.     Anesthetize  quickly  with  ether  (do  not  use  chloro- 
form), insert  a  canula  in  the  carotid,  bleed  completely,  defibrinate  the  blood 
and  dilute  it  with  nine  volumes  of  Locke's  solution.     Use  this  diluted  blood 
as  a  normal  solution  for  perfusing  the  heart.     Reserve  enough  of  the  solution 
for  making  the  drug  mixtures,  pour  the  remainder  into  the  perfusion  appara- 
tus described  on  page  73,  fill  the  tubes,  adjust  the  apparatus  and  bring  to  a 
constant  temperature  of  36  to  37°  C.  (a  higher  temperature  is  unfavorable). 
Quickly  remove  the  heart,  taking  care  only  to  preserve  enough  of  the  aorta 
for  the  insertion  of  the  canula  without  danger  of  interfering  with  the  semi- 
lunar  valves.     Mount  the  heart  without  catching  air  in  the  canula,  attach  the 
recording  lever  of  the  Guthrie  cardiograph  and  start  the  perfusion.     The 
perfusion  pressure  should  be  from  80  to  100  cm.  of  water. 

The  heart  contracts  and  a  uniform  rhythm  will  be  quickly  established 
and  may  be  maintained  for  several  hours.  Perfuse  the  heart  with  0.2 
percent  alcohol  in  the  Locke-blood  solution  for  from  30  to  100  seconds 
at  a  time,  allowing  full  time  for  a  return  to  the  normal  after  each  drug 
perfusion.  Raise  the  dose  successively  to  0.4  percent,  i  percent,  and  2 
percent  of  alcohol.  The  stronger  solutions  reduce  the  amplitude  and 
ultimately  the  rate  of  the  heart;  the  weaker  doses,  according  to  Dixon,  in- 
crease the  amplitude. 

5.  Alcohol  on  muscular  work.     Ligate  one  leg  of  a  frog  near  the 
thigh  to  exclude  its  circulation  (a  quarter-inch  rubber  tube  makes  a  fine 
ligature  for  this  purpose).     Inject  0.3   c.c.   (5  minims)   of    95  percent 
alcohol  into  the  dorsal  lymph  sac.     In  exactly  twenty  minutes  pith  the 
frog,  pin  it  out  on  the  frog-board  with  the  ventral  side  down,  and  ligate  the 
alcoholized  leg.     Quickly  prepare  the  tendon  of  the  normal  muscle,  keeping 
the  muscle  covered  with  skin,  attach  to  the  muscle  lever,  and  determine  the 
work  it  can  do  by  stimulating  the  muscle  directly  with  a  single  induction 
shock  once  every  two  seconds  until  the  muscle  is  completely  exhausted. 
Load  the  muscle  with  a  30-  to  50-  gram  weight.     Record  the  contractions  on 
the  lower  half  of  a  drum  with  a  speed  of  i  mm.  a  second. 


4  EXPERIMENTAL    PHARMACOLOGY. 

Prepare  the  second  or  alcoholized  muscle  just  twenty  minutes  after  it 
has  been  ligated,  mount,  load,  and  stimulate  in  the  same  manner.  Record 
the  second  experiment  on  the  upper  half  of  the  same  smoked  paper  and 
parallel  with  the  record  of  the  first.  Repeat  this  experiment  using  a  dose 
of  i  c.c.  of  95  percent  alcohol,  to  demonstrate  the  injurious  effects  of 
alcohol  on  the  work  of  the  muscle.  (Lee  and  Salant,  Am.  Jour.  Physiology, 
Volume  VIII,  p.  61,  1902.) 

A  more  difficult  but  more  accurate  experiment  is  obtained  as  follows: 
Destroying  the  brain  only  of  a  frog,  pin  it  to  the  frog-board  belly  down, 
dissect  out  the  right  tendon  Achilles,  and  attach  to  a  muscle  lever.  Isolate 
the  lumbar  plexus  on  the  right  of  the  urostyle,  using  care  not  to  interfere 
with  the  circulation  of  the  gastrocnemius.  Stimulate  the  right  muscle  as 
above.  Now  inject  alcohol  into  the  abdominal  cavity,  and  after  20  to  30 
minutes  of  absorption  measure  the  work  of  the  left  gastrocnemius.  This 
method  has  the  advantage  of  maintaining  the  circulation  intact  for  both  the 
normal  and  the  alcoholized  muscle. 

The  effect  of  alcohol  on  the  speed  of  the  contraction  can  be  determined 
by  one  of  two  methods.  One  can  measure  the  simple  muscle  contraction 
before  and  after  alcoholization.  Perhaps  a  better  method  is  to  use  Lee's 
automatic  stimulating  device  (Am.  Jour.  Physiol.,  VIII,  p.  61)  which  auto- 
matically stimulates  a  muscle  at  the  moment  of  complete  relaxation.  Re- 
cord the  contractions  on  a  slowly  moving  drum,  2  mm.  per  second. 

6.  Alcohol  on  voluntary  work  of  human  muscle.    Demonstration. 
Measure  the  voluntary  power  of  the  flexors  of  the  middle  finger  with  a  load 
of  three  kilos  or  more,  using  Mosso;s  ergograph.     Take  two  or  three  normal 
records  of  voluntary  contractions  at  intervals  of  20  minutes.     Now  take  a 
dose  of  20  to  40  c.c.  of  20  percent  alcohol,  according  to  the  susceptibility 
of  the  individual.     Remeasure  the  muscular  power  after  60,  90,  and  120 
minutes,  respectively.     Compute  the  work  done  in  kilogrammeters.    (Lom- 
bard, Jour.  Physiol.,  Vol.  13,  p.  49;  Hallsten,  Skand.  Arch.  f.  Physiol.  Bd. 
16.   S.   139.) 

7.  Alcohol  on  the   circulatory  and  respiratory  systems  of  the 
mammal.     Anesthetize    a    dog    with    morphine    and    chloroform,    p.  70. 
Take  the  blood-pressure  from  the  carotid  artery,  and  the  respiration  from  a 
side  branch  of  a  tracheal  canula.     Expose  the  saphenous  vein  .and  insert 
a  canula  for  intravenous  injections,  and  attach  it  to  a  50  c.c.  buret. 

Students  who  have  attained  the  requisite  skill  should  take  an  onkomet- 
ric  record  with  the  blood-pressure.  To  prepare  for  this  record  open  the 
abdominal  cavity  of  the  dog,  remove  the  outer  sheath  from  the  left  kidney 


ALCOHOL.  5 

and  enclose  that  organ  in  a  renal  onkometer.  Record  the  kidney  volume 
changes  by  means  of  a  Brodie's  bellows,  or  Roy's  piston  recorder,  page  71. 

The  anesthetic  must  be  given  with  perfect  regularity,  2  to  6  drops  of 
chloroform  every  30  seconds,  the  exact  amount  that  will  maintain  constant 
anesthesia  to  be  quickly  ascertained  for  each  animal. 

Take  a  record  on  the  continuous  kymograph  and,  when  all  is  in  good 
working  condition  and  a  normal  record  has  been  secured,  slowly  inject  20 
percent  warmed  alcohol  from  the  buret  into  the  vein  until  some  decided 
effect  on  the  blood-pressure  is  noted,  i.e.,  after  a  dose  of  20  c.c.  or  more. 
Extreme  caution  must  be  observed  lest  the  heart  by  rapid  perfusion  be  sub- 
jected to  an  overconcentrated  solution.  The  experiment  should  be  re- 
peated with  different  doses. 

Since  the  anesthetics  used  all  depress  the  irritability  of  the  circulatory 
apparatus,  this  experiment  ought  to  be  demonstrated  on  a  decerebrate  ani- 
mal. In  such  an  animal  the  medulla  being  intact  will  maintain  natural 
respirations.  Any  alcoholic  stimulation  of  the  medullary  centers  can  easily 
be  observed.  The  recommended  intravenous  dose  of  alcohol  will  produce 
slowing  of  the  heart,  a  phenomenon  which  disappears  on  section  of  the  vagi, 
thus  indicating  a  direct  effect  on  the  vagal  centers. 

8.  Alcohol  on  the  reaction  time  of  the  reflex  frog.  Destroy  the 
brain  of  a  frog,  including  the  medulla,  and  when  it  has  recovered  from  the 
shock  test  the  normal  reaction  time  to  electrical  stimuli  applied  to  the  toe. 
Measure  the  time  of  the  reaction  with  a  watch,  or  record  it  with  a  writing- 
point  attached  to  the  foot  or  leg  of  the  suspended  frog.  Give  a  dose  of  0.3 
c.c.  (5  minims)  of  95  percent  alcohol  in  the  dorsal  lymph  sac.  Retest 
the  reaction  time  at  exactly  20  and  40  minutes  after  the  injection.  Com- 
pare the  results  with  experiments  i  and  5  above. 


EXPERIMENTAL    PHARMACOLOGY. 


ETHER. 

List  of  Experiments  Illustrating  the  Effects  of  Ether.  PAGE. 

1.  On  the  frog 6 

2.  On  the  ventricular  muscle 6 

3.  On  the  frog's  heart 7 

4.  On  the  mammalian  heart 7 

5.  On  the  irritability  of  voluntary  muscle 7 

6.  On  the  irritability  of  nerve  tissue 9 

7.  On  the  blood-pressure  and  respiration  of  a  mammal     .    .  9 

8.  On  the  germination  of  seeds 10 

9.  On  the  growth  of  yeast n 

1.  Ether  on  the  frog.     Inject  0.2  c.c.  (31/2  minims)  of  ether1  into 
the  dorsal  lymph  sac  or  the  abdominal  cavity  of  a  frog.     Give  0.3  c.c.  to  a 
second  frog.     The  dose  can  be  given  more  accurately  from  the  hypodermic  if 
a  50  percent  solution  of  ether  in  olive  oil  is  used.     The  first  dose  will  produce 
anesthesia  in  about  10  minutes.     The  stages  most  readily  observed  are :     ist, 
great   excitement   shown   by   rapid   respirations,    active   movements,    and 
increased  reflex  irritability;    2d,  slower  respirations,  very  sluggish  response 
to  external  stimulation;  3d,  loss  of  voluntary  muscular  control  and  sometimes 
of  respiratory  motions.     Slight  power  of  reflex  response  is  retained,  including 
eye  reflexes.     The  voluntary  motions  will  be  regained  in  from  60  to  90 
minutes  if  the  animal  is  kept  moist  (winter  frogs),  and  complete  recovery 
in  two  hours.     The  frog  will  recover  from  the  larger  dose  in  from  20  to  24 
hours,  or  it  may  even  fail  of  recovery. 

2.  Ether  on  the  ventricular  muscle.    Mount  a  strip  of  terrapin's 
ventricle  and  establish  rhythmic  contractions  in  a  bath  of  0.7  percent  saline. 
Record  on  a  drum  moving  i  to  2  mm.  per  second.     Immerse  the  strip  in  a 
bath  of  i  percent  ether  in  saUne  for  two  to  three  minutes,  then  return 
to  the  physiological  saline  bath.     The  sharp  decrease  in  both  amplitude 
and  rate  of  contractions  is  recovered  quickly  in  the  saline  bath. 

Repeat  the  experiment  using  2,  4,  and  6  percent  ether  solutions. 
The  weaker  solutions  occasionally  produce  slight  but  temporary  increase 
in  the  rate,  the  initial  excitation  stage.  Also  use  strips  of  auricle  and  sinus. 

JThe  dose  is  figured  for  a  40-gram  frog.  Proportionate  doses  should  be  given  for  other 
weights.  In  all  experiments  on  frogs  that  depress  their  functions,  the  animal  should  be  retained 
in  a  moist  bell  jar  for  as  much  as  24  hours,  if  necessary,  in  order  to  test  the  animal's  power  of 
recovery. 


ETHER.  7 

3.  Ether  on  the  frog's  heart.     Pith  a  frog,  expose  its  heart,  insert 
a  canula  in  the  inferior  vena  cava  and  perfuse  the  heart  in  place  by  the 
method  described  for  alcohol,  experiment  3.    This  brings  the  solution  into 
intimate  contact  with  the  entire  heart  and  it  responds  almost  instantly  to 
any  change  in  the  composition  of  the  irrigating  fluid.     Perfuse  the  heart 
first  with  Ringer's  solution  and  follow  with  i  percent  ether  in  Ringer's 
solution. 

4.  Ether  on  the  mammalian  heart.     Use  the  Roy-Adami  method 
(given  by  Cushny,   Jour.   Exp.   Medicine,   Volume  II,  page  233)   or  the 


FIG.  2. — The  action  of  ether  on  isolated  heart  muscle.  In  this  experiment  a  strip 
of  terrapin's  ventricle  was  mounted  in  physiological  saline  until  a  regular  rhythmic 
beat  was  established.  The  saline  was  then  drawn  off  and  the  strip  left  suspended  in 
moist  air.  At  the  point  indicated  ether  vapor  was  driven  through  the  moist  chamber 
until  the  contractions  ceased.  The  ether  vapor  was  then  removed  with  moist  air 
at  the  second  arrow  and  the  recovery  of  the  rhythm  took  place  as  shown. 

method  described  on  page  73  and  used  in  experiment  4  of  the  alcohol 
series.  Etherize  a  cat  or  rabbit,  draw  the  blood,  defibrinate  it,  and  dilute 
it  with  nine  volumes  of  Locke's  solution  and  use  as  a  standard  Locke-Blood 
perfusion  fluid.  Remove  the  heart  and  adjust  it  in  the  apparatus,  page  74. 
Perfuse  with  the  Locke-Blood  solution  and  when  the  rhythm  is  established 
change  to  i  percent  ether  in  Locke-Blood.  Use  2  percent  ether  in  a 
later  experiment. 

5.  Ether  on  the  irritability  of  voluntary  muscle.  Mount  a  gas- 
trocnemius  of  the  frog  in  the  moist  chamber,  arrange  to  stimulate  the 
muscle  directly  with  a  current  of  medium  intensity  but  which  produces  a 
maximal  contraction.  Adjust  a  vapor  apparatus  containing  saturated 


EXPERIMENTAL    PHARMACOLOGY. 


S.S 


2  o 

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ex  o 


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M  en 
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en  oj 

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D    C 


§'5 


ETHER.  9 

ether  water  ready  for  quick  connection  with  the  gas  tube  of  the  moist 
chamber.  Stimulate  the  muscle  with  single  induction  currents  once  every 
30  seconds  throughout  the  entire  experiment,  whether  contractions  are 
secured  each  time  or  not.  Record  on  a  drum  having  a  rate  of  2  mm.  per 
10  seconds. 

Take  records,  three  or  four  normal  contractions,  then  turn  on  the  ether 
vapor  for  five  minutes.  Quickly  remove  the  vapor  with  a  current  of  fresh 
air.  The  muscle's  irritability  will  decrease  to  a  point  at  which  the  stimulus 
is  submaximal  or  even  subminimal,  but  when  the  ether  vapor  is  removed 
the  contractions  quickly  reappear  and  attain  their  former  amplitude.  Use 
small  muscles  for  this  experiment. 


FIG.  4. — The  action  of  ether  on  isolated  strips  from  the  auricle  (upper  trace)  and 
from  the  sinus  (lower)  of  the  terrapin.  The  strips  were  giving  tone  contraction  waves, 
but  no  fundamental  rhythm  when  bathed  with  normal  sodium  chloride  solution.  At  the 
mark  "on"  the  sodium  chloride  was  changed  to  one  percent  ether  in  normal  saline 
for  seven  minutes,  then  again  to  the  normal.  During  the  ether  bath  the  tone  waves 
disappear,  a  condition  which  lasts  for  three  minutes  after  the  ether  is  removed.  Fol- 
lowing the  ether  there  is  a  renewal  of  the  tone  waves  which  are  even  more  rapid  than 
in  the  normal.  Time  in  minutes. 

One  may  with  this  preparation  also  demonstrate  that  the  muscle  has  a 
diminished  power  to  do  work  when  etherized,  method  page  72. 

6.  Ether  on  the  irritability  of  nerve   tissue.     Prepare  a  muscle 
nerve  of  the  frog,  isolating  the  entire  sciatic  with  a  piece  of  cord,  and  with  the 
skin  covering  the  muscle.     Mount  the  preparation  with  the  nerve  in  the 
moist  chamber  and  on  the  electrodes,  but  with  the  muscle  hanging  through 
the  hole  in  the  floor  of  the  moist  chamber  and  on  the  outside  so  that  it  will 
not  be  etherized.     Close  the  hole  with  a  sheet  of  moist  filter-paper.     Proceed 
exactly  as  in  experiment  5  above  testing  the  irritability  of  the  nerve  through 
its  effect  on  the  muscle. 

The  influence  of  ether  on  nerve  irritability  may  also  be  demonstrated 
directly  from  the  nerve  by  the  action  current  method.  For  a  description  of 
the  method,  see  Am.  Jour.  Physiol.,  Volume  I,  p.  104. 

7.  Ether  on  the  blood-pressure  and  on  the  respiration  of  the 
mammal.     Anesthetize  a  dog  with  morphine  and  ether.     Introduce  an 


10  EXPERIMENTAL    PHARMACOLOGY. 

arterial  canula  in  the  carotid  for  taking  the  blood-pressure  record,  see  page 
70  for  details  of  method.  Insert  a  tracheal  canula  and  take  a  record  of  intra- 
tracheal  pressure  from  a  T-tube  attached  to  the  canula.  Give  ether  from 
an  ether  bottle  connected  to  the  end  of  the  tracheal  tube.  Take  a  continuous 
record  and  ultimately  give  excess  of  ether,  then  allow  partial  and  guarded 
recovery.  Give  ether  to  the  point  where  respirations  cease,  a  point  attained 
with  difficulty  except  when  the  animal  has  a  large  dose  of  morphine.  The 
blood-pressure  is  an  index  of  safety,  for  it  has  been  shown  that  respiratory 
impulses  are  quickly  re-established  when  the  blood-pressure  remains  high. 

Saturated  ether  in  saline  as  an  intravenous  injection  in  doses  of  20  c.c. 
and  more,  given  along  with  a  uniform  administration  of  ether  by  the  trachea, 


FIG.  5. — The  influence  of  ether  vapor  upon  the  irritability  of  the  sciatic  nerve 
in  the  muscle-nerve  preparation  of  a  frog.  The  muscle-nerve  preparation  was  sus- 
pended in  a  moist  chamber  with  the  muscle  hanging  through  the  opening  in  the  cham- 
ber. The  first  two  contractions  are  normal.  The  next  eight  are  successive  contrac- 
tions at  intervals  of  ten  seconds  during  the  passing  of  ether  vapor.  At  the  X  the 
ether  vapor  was  removed  by  a  stream  of  moist  air,  the  nerve  being  stimulated  at  inter- 
vals of  ten  seconds  until  recovery  of  the  irritability  as  shown  by  the  contractions  at  the 
last  part  of  the  experiment.  A  gap  of  two  minutes  occurs  at  X,  during  which  the  nerve 
was  not  irritable.  Time  in  seconds. 

will  often  demonstrate  the  characteristic  circulatory  and  respiratory  effects 
of  the  drug. 

The  rectal  temperature  should  be  recorded  at  intervals  to  demonstrate 
the  fall  of  temperature  under  anesthetics.  Note  also  the  state  of  dilatation 
of  the  pupil. 

8.  Ether  on  the  germination  of  seeds.  Arrange  two  eight-ounce 
wide-mouth  bottles  with  stoppers  each  fitted  with  two  glass  tubes,  letting 
one  tube  extend  to  near  the  bottom  of  the  bottle.  Suspend  in  each,  by 
means  of  cotton,  a  dozen  seeds — corn,  wheat,  clover,  beans,  etc. — and  in- 
troduce just  enough  water  to  maintain  a  saturated  vapor.  Set  both  bottles  in 
a  window.  Twice  a  day  for  a  week,  pass  through  one  saturated  ether 


CHLOROFORM.  II 

vapor,  through  the  other  air.  The  seeds  in  both  will  swell  from  the  ab- 
sorption of  the  water,  but  only  the  seeds  in  the  bottle  with  pure  air  intro- 
duced will  grow.  Reverse  the  two.  The  sprouting  grain  will  have  its 
growth  checked  and  the  etherized  seeds  will  begin  to  grow. 

9.  Ether  on  the  yeast.  Take  two  fermentation  tubes  of  active  yeast 
culture,  add  2  c.c.  pure  ether  to  one.  Note  the  relative  rate  of  gas  liberation. 

CHLOROFORM. 

List  of  Experiments  Illustrating  the  Effect  of  Chloroform.  PAGE. 

1.  On  the  frog n 

2.  On  the  heart  strip n 

3.  On  the  frog's  heart .  .    .  n 

4.  On  the  mammalian  heart 12 

5.  On  muscular  irritability 12 

6.  On  nerve  irritability 12 

7.  On  the  blood-pressure  and  respiration  of  a  mammal.    ...  12 

8.  On  the  kidney  secretion 14 

9.  On  germinating  seeds 14 

1.  Chloroform  on  the  frog.     Inject  0.06  c.c.  of  pure  chloroform 
or  0.3  c.c.  of  20  percent  chloroform  in  olive  oil  into  the  dorsal  lymph 
sac  or  into  the  abdominal  cavity  of  a  frog.     The  anesthesia  is  more  pro- 
found and  the  recovery  less  rapid  than  in  the  case  of  ether.     Determine  the 
relative  intensity  of  action  of  chloroform  and  ether  by  your  own  experiments. 

2.  Chloroform  on  the  heart  strip.    Proceed  as  with  ether  in  ex- 
periment 2,  page  6,  using  a  0.05  percent  solution  of  chloroform  in 0.7  percent 
saline  for  one  to  three  minutes.     The  contractions  cease  almost  at  once. 
Recovery  in  saline  takes  place  very  slowly.     In  comparison  with  ether  the 
period  of  anesthesia  is  long.     The  amplitude  of  the  first  contractions  to 
reappear  is  very  slight  and  the  recovery  rate  slow  and  irregular.     The 
original  character  of  activity  is  not  restored  within  20  to  40  minutes.     Re- 
peat using  a  solution  of  o.i  percent  chloroform. 

An  instructive  picture  is  given  by  parallel  records  of  experiments  on 
strips  from  the  same  heart  showing  the  effects  of  4  percent  ether  and  of 
o.i  percent  chloroform  for  two  minutes,  both  in  saline. 

3.  Chloroform  on  the  frog's  heart.     Proceed  as  in  the  similar  ex- 
periment with  ether,  page  7,  using  0.5  percent  chloroform  in  physiolog- 
ical saline  to  irrigate  the  outer  surface  of  the  heart.     Or  perfuse  the  heart 


12 


EXPERIMENTAL    PHARMACOLOGY. 


s 


cj 


with  0.05  percent  chloroform  in  saline 
through  the  vena  cava.  Care  must  be 
used  not  to  prolong  the  action  of  the  drug. 
The  amplitude  is  reduced  to  one-half  and 
the  rate  markedly  slowed  or  entirely  sup- 
pressed. Both  rate  and  amplitude  are 
recovered,  but  not  so  quickly  as  with  ether. 

4.  Chloroform  on  the  mammalian 
heart.     Determine  the  action  of  chloroform 
on  the  isolated  cat's  heart,  using  the  method 
described    for   the    alcohol    experiment   4, 
page  3.     Perfuse   the   heart  with  chloro- 
form 0.02  to  0.05  percent  in  the  Locke- 
blood.     The  chloroform  perfusion  must  be 
for  short  periods  and  be  guarded  closely. 

5.  Chloroform    on    the    muscular 
irritability.     See  ether  experiment  5,  page 
3.     Use  o.i   percent  chloroform  water 
in    the   vapor  apparatus.      Care  must  be 
taken  to  remove  the  saturated  chloroform 
vapor  from  the  vapor  apparatus  just  before 
using,  otherwise  the  muscle,  or  the  nerve 
in  the  next  experiment,  will  be  over  anes- 
thetized and  will  not  recover  its  irritability. 
There  is  no  danger  with  ether  from  this 
cause. 

6.  Chloroform  on  the  irritability  of 
the   nerve.     Repeat   ether   experiment  6. 
Use  o.i  percent  chloroform  water  in  the 
vapor  apparatus. 

7.  Chloroform      on      the      blood- 
pressure  and  on  the  respiration  of  a 
mammal.     Proceed  as  with  ether  experi- 
ment 7,  page  4,  using  chloroform  to  anes- 
thetize  the   dog   or   cat    (rabbits   are    too 
sensitive  to  chloroform  for  use  in  this  ex- 
periment except  in  practiced  hands).     Re- 
member   that    chloroform    is   said    to   be 
about  forty  times  as  strong  as  ether  in  its 


CHLOROFORM.  13 

general  effects  on  the  animal  body.  If  the  vagi  are  intact  and  the  animal 
is  anesthetized  without  tracheotomy  there  will  be  marked  slowing  of  the 
heart  rate  together  with  a  sharp  fall  of  blood-pressure.  This  effect  is 


FIG.  7. — Chloroform  perfusion  of  the  frog's  heart.  Ringer's  solution  used  for  the 
normal,  o.i  percent  chloroform  between  "on-"  and  "off. "  Perfusion  pressure 
4  cm.  Record  from  the  suspended  apex.  Time  in  seconds. 

eliminated  by  section  of  the  vagi.  With  extreme  care  chloroform  anesthesia 
may  be  pushed  to  the  point  where  respirations  cease,  and  the  animal  be 
recovered  without  artificial  respiration.  Often,  however,  in  5  to  10  seconds 


FIG.  8. — The  effect  of  chloroform  on  the  irritability  of  the  nerve  in  the  muscle- 
nerve  preparation.  The  first  two  contractions  represent  the  normal  amplitude  upon 
stimulating  the  nerve  of  a  preparation  mounted  in  a  moist  chamber.  The  muscle  was 
allowed  to  hang  through  the  opening  in  the  floor  of  the  moist  chamber,  so  as  to  protect 
it  from  chloroform  vapor.  At  the  point  indicated  by  the  word  "on"  chloroform  vapor 
was  driven  through  the  moist  chamber.  The  nerve  was  stimulated  at  regular  inter- 
vals until  no  further  contractions  occurred.  The  chloroform  was  next  removed 
with  moist  air  and  the  stimulations  continued.  After  a  short  interval  contractions 
gradually  resumed  until  they  reached  their  normal  maximum. 

after  respirations  cease,  the  blood-pressure   will  suddenly  sink  to  a  low 
level,  and  the  heart  will  become  weak  and  slow  (see  experiment  8  below), 


14  EXPERIMENTAL    PHARMACOLOGY. 

a  state  from  which  recovery  can  be  secured  only  by  rapid  and  vigorous 
artificial  respiration. 

Give  chloroform  intravenously  in  doses  of  10  to  20  c.c.  of  0.5  percent 
solution  in  saline,  allowing  plenty  of  time  for  recovery  in  each  test.  Com- 
pare with  alcohol  and  ether. 

8.  Chloroform  on  the  secretion  of  the  kidney.    Anesthetize  a  dog 
with  morphine,  i  c.c.  of  i  percent,  and  chloroform,  avoiding  deep  anesthesia 
during  the  preliminary  preparations.     Insert  a  ureter  canula  and  connect 
it  with  a  horizontal  glass  tube  mounted  on  a  graduated  scale,  or  see  methods, 
page  70.     Take  a  continuous  record  of  the  arterial  pressure. 

Now  determine  the  normal  rate  of  secretion  of  urine  per  10  minutes 
for  at  least  40  minutes,  keeping  the  dog  under  light  but  constant  anesthesia. 
Inject  intravenously  10  c.c.  of  0.5  percent  chloroform  solution  in  saline. 
Double  the  dose  if  necessary  until  profound  anesthesia  with  low  blood- 
pressure  and  weak  heart  is  obtained.  Or  produce  deep  anesthesia  by  means 
of  the  respiratory  inhalations.  Recover  and  maintain  light  anesthesia  for 
an  hour  or  more.  The  circulation  is  quickly  re-established  in  good  condi- 
tion, but  the  secretion  of  urine  which  is  suppressed  during  the  stage  of 
deep  anesthesia  is  more  slowly  brought  up  to  the  normal  with  the  re-establish- 
ment of  good  circulation. 

9.  Chloroform    on    germinating    seeds.     Repeat    the    experiment 
described  for  ether,  page  n,  passing  air  from  saturated  chloroform  water  into 
one  bottle  of  seed,  and  pure  air  into  the  other.     After  the  seeds  in  air  have 
sprouted,  reverse  the  bottles.     Both  seeds  and  young  growing  plants  are 
anesthetized  by  chloroform.     The  seeds  may  not  grow  later,  as  the  drug  kills 
plant  protoplasm  when  given  beyond  a  rather  narrow  limit  of  both  time 
and  concentration. 

CHLORAL  HYDRATE. 

Experiments  Showing  the  Effects  of  Chloral  Hydrate.  PAGE. 

1.  On  the  frog 14 

2.  On  the  rabbit  or  cat 15 

3.  On  the  heart  of  the  frog 15 

4.  On  the  heart  muscle.  15 

i.  Chloral  hydrate  on  the  frog.  Give  a  hypodermic  injection  of 
0.5  c.c.  of  2  percent  chloral  hydrate  dissolved  in  0.7  percent  saline. 
Keep  the  animal  in  a  moist  battery  jar  until  complete  recovery.  Give 
particular  attention  to  the  effects  on  the  circulatory  and  the  nervous  systems. 


THE    OPIUM    SERIES.  15 

2.  Chloral  on  the  rabbit  or  cat.     Give  a  hypodermic  injection  of 
2  c.c.  of  2  percent  chloral  hydrate  per  kilo  of  body  weight  In.  saline. 
Repeat  in  sixty  minutes  if  necessary  to  produce  the  chloral  narcosis.    Make 
close  comparison  with  the  effect  of  morphine  and  strychnine. 

3.  Chloral  hydrate  on  the  frog's  heart.     Pith  a  frog  and  take  trac- 
ings of  the  ventricle  when  irrigated  over  the  surface  with  i  percent  chloral 
hydrate.     Or  perfuse  the  heart  with  0.2   percent  chloral  hydrate  in 
Ringer's  solution  and  take  tracings  of  the  ventricle  by  the  method  described 
on  page  69.     Note  that  the  recovery  period  is  unusually  long. 

4.  Chloral  hydrate  on  heart  muscle.    Prepare  a  ventricular  strip 
of  the  terrapin  and  establish  contraction  in  a  bath  of  0.7  percent  sodium 
chloride  as  usual.     Change  to  a  bath  of  o.i  percent  chloral  hydrate 
in  sodium  chloride.     Stronger  solutions  may  suppress  the  rhythm  entirely. 

THE  OPIUM  SERIES. 

Experiments  Illustrating  the  Effects  of  Morphine,  Codeine  and  The- 
baine.  PAGE. 

1.  On  the  frog 15 

2.  On  the  mammal 15 

3.  On  ventricular  muscle 16 

4.  On  the  frog's  heart 18 

5.  On  the  mammalian  heart 18 

6.  On  the  reflex  reaction  time 18 

7.  On  the  volume  of  air  breathed 18 

8.  The  morphine  group  on  the  circulation  and  respiration  in 

the  mammal 18 

1.  Morphine  on  the  frog.     Give  a  dose  of  i  c.c.  of  2  percent  mor- 
phine acetate  in  physiological  saline  in  the  dorsal  lymph  sac.     Keep  the 
animal  under  observation  for  two  or  more  hours  to  secure  the  later  effects 
in  the  frog.     An  instructive  comparison  is  had  by  giving  a  dose  of  0.5  c.c. 
(8  minims)  of  o.i  percent  strychnine  nitrate  to  a  second  frog  at  the  same 
time.     Keep  the  frog  under  observation  until  complete  recovery. 

Give  a  second  frog  an  injection  of  i  c.c.  of  i  percent  codeine  in  phy- 
siological saline.     Compare  with  morphine. 

The  dose  of  thebaine  for  the  frog  is  i  c.c.  of  i  percent. 

2.  Morphine  on  the  mammal.     Give  a  hypodermic  injection  of  1.5 
c.c.  of  2  percent  morphine  under  the  skin  of  the  shoulder,  see  anes- 


i6 


EXPERIMENTAL    PHARMACOLOGY. 


thesia,  p.  65.  Keep  the  animal 
under  observation  for  at  least 
two  hours  and  note  at  intervals 
the  temperature,  irritability, 
respiratory,  ocular,  and  other 
changes  under  the  influences  of 
the  drug.  If  vomiting  is  pro- 
duced a  concentrated  watery  ex- 
tract of  the  vomit  will  show  the 
presence  of  morphine,  thus 
demonstrating  that  excretion  of 
morphine  takes  place  into  the 
stomach. 

3.  Morphine  on  the  ven- 
tricular muscle.  Prepare  a 
strip  of  the  ventricle  of  a  terrapin 
and  get  it  into  regular  contrac- 
tions by  a  bath  of  0.7  percent 
sodium  chloride.  Change  the 
strip  to  a  bath  of  i  percent 
morphine  in  sodium  chloride 
for  five  minutes,  or  until  a 
marked  change  in  the  rate  and 
amplitude  of  the  contractions 
occurs,  after  which  the  strip 
should  be  returned  to  saline. 
Record  the  contraction  on  a 
smoked  drum  moving  with  a 
speed  of  i  to  2  mm.  a  second. 
This  experiment  shows  that  the 
contractions  of  cardiac  muscle 
are  weakened  and  slowed  under 
morphine.  Repeat,  varying  the 
conditions  according  to  the  re- 
sults of  the  previous  experiment. 
A  stronger  solution  of  morphine 
will  inhibit  all  heart  muscle 
activity  for  many  minutes  or 
even  hours.  The  proper 
strength  of  codeine  for  this 


THE    OPIUM    SERIES. 


1 8  EXPERIMENTAL    PHARMACOLOGY. 

experiment  is  0.5  percent  in  physiological  saline,  of  thebaine  about  i 
percent  solution. 

4.  Morphine  on  the  frog's  heart.     Pith  a  frog,  expose  the  heart 
and  take  a  record  of  its  contractions,  either  by  the  perfusion  or  by  the  sus- 
pension method.     Test  the  irritability  of  the  vagus  trunk.     If  the  circulation 
is  still  effective  give  a  lymph  sak:  injection  of  0.5  c.c.  of  10  percent  mor- 
phine acetate  or  apply  drops  of  this  solution  directly  to  the  heart  from  the 
dropping  bottle.     Retest  the  effectiveness  of  the  vagus  stimulation.     If  the 
heart  is  perfused,  the  effective  solution  to  use  is  0.5.  percent  of  morphine 
acetate  in  Ringer's  solution.  ,; 

5.  Morphine  on  the  mammalian  heart. — Etherize  a  cat,  collect  its 
blood  and  isolate  its  heart  as  described  in  alcohol  experiment  4.     Fill  the 
tubes  of  the  mammalian  heart  perfusion  apparatus,  bring  the  apparatus  to  a 
temperature  of  36°  C.,  adjust  the  heart  in  it  and  begin  the  normal  perfusion. 
When  the  heart  is  contracting  with  a  regular  and  relatively  even  rhythm 
perfuse  it  for  two  minutes  with  0.5  percent  morphine  in  Locke-blood  solu- 
tion.    Repeat  the  experiment  using  i  percent  solution.     If  the  perfusion 
is  too  prolonged  the  heart  depression  will  be  removed  only  with  the  greatest 
difficulty. 

6.  Morphine  on  the  reflex  reaction  time.     Test  the  reaction  time 
in  a  reflex  frog  in  the  usual  way,  see  methods  page  71,  first  on  the  normal 
animal,  then  20  and  40  minutes  after  a  dose  of  i  c.c.  of  4  percent  morphine 
acetate. 

7.  Morphine  on  the  volume  of  air  breathed.    Anesthetize  a  rabbit 
(or  cat)  with  two  grams  urethane  and  ether,  administering  the  latter  at 
perfectly  regular  intervals  and  with  a  constant  number  of  drops.     Keep  a 
continuous  record  of  the  respiration  rate  per  minute  either  by  counting  or  by 
recording  on  a  smoked  drum.     Measure  the  respiration  volume  as  follows: 
Insert  a  tracheal  canula  and  connect  it  with  an  apparatus  for  measuring  the 
volume  of  expired  air.     Ether  can  be  given  through  the  open  tube  of  the 
apparatus  except  when  actually  measuring  the  air  volume.     Fill  the  grad- 
uated cylinder  with  water.     Then  measure  the  volume  of  eight  or  ten  expi- 
rations, according  to  the  volume  of  the  apparatus  used,  and  repeat  several 
times  to  secure  reliable  averages  of  the  expiratory  volume  of  the  etherized 
animal.     Compute  the  expiratory  volume  per  minute.     Now  give  i  c.c.  of 
2  percent  morphine   hypodermic.     Remeasure  the  respiratory  rate  and 
expiration  volume  at  intervals  of  10  minutes. 

8.  The  morphine  group  on  the  circulation  and  on  respiration  in 
the  mammal.     Anesthetize  a  dog  with  chloroform  (no  morphine),  take  the 


THE    OPIUM    SERIES.  1 9 

blood-pressure  from  the  carotid,  insert  a  tracheal  canula  and  take  the 
respiration  by  the  intratracheal  method.  Insert  a  canula  into  the  saphen- 
ous  vein  and  connect  with  a  buret  for  intravenous  injections.  (One  may 
readily  insert  a  canula  into  the  ureter  and  follow  the  secretion  of  urine 
under  the  morphine.  Consult  the  instructor.) 

Give  an  intravenous  injection  of  2  c.c.  of  2  percent  morphine  in  saline. 
The  injected  solution  should  be  about  the  temperature  of  the  body.  Repeat 
after  ten  minutes,  using  4  c.c.  When  equilibrium  is  reestablished  give 
2  c.c.  of  i  percent  codeine.  Give. 2  c.c.  of  i  percent  thebaine.  Give 
thebaine  first  if  there  is  an  opportunity  to  make  the  test  on  a  second  animal. 
Now  cut  the  vagus  nerves  and  repeat  the  dose  of  2  c.c.  of  2  percent 
morphine. 

The  anesthetic  must  be  gradually  diminished  according  to  the  amount 
of  morphine,  etc.,  that  has  been  injected.  Use  artificial  respiration  if 
necessary. 


2O  EXPERIMENTAL  PHARMACOLOGY. 


CAFFEINE. 

Experiments  Illustrating  the  Effects  of  Caffeine.  PAGE. 

1.  On  the  frog 20 

2.  On  the  ventricular  muscle 20 

3.  On  the  frog's  heart 20 

4.  On  muscular  irritability  and  on  muscular  work 21 

5.  On  voluntary  work  of  human  muscle 21 

6.  On  the  reaction  time  in  man  .    .    . 22 

7.  On  the  reflex  reaction  time  in  the  frog 22 

8.  On  the  mammalian  heart 23 

9.  On  the  circulation  and  respiration  in  the  mammal     ...  23 
10.  As  a  diuretic       24 

1.  Caffeine  on  the  frog.     The  dose  is  i<c.c.  of  0.5  percent  caffeine 

in  the  dorsal  lymph  sac.  There  is  usually  a  great  increase  in  the  irritability 
together  with  muscular  cramps  in  the  later  stages,  and  finally  paralysis. 
There  may  be  considerable  opisthotonus  from  the  direct  muscular  effects  at 
the  area  of  injection.  Note  the  recovery  stages  when  the  frog  is  kept  in  a 
moist  battery  jar. 

2.  Caffeine  on  the  ventricular  muscle.     Record  the  contractions  of 
a  strip  of  terrapin's  ventricle  beating  in  physiological  saline  on  a  slow-speed 
drum.     Change  the  strip  from  saline  to  o.i  percent  caffeine  in  saline  for 
five  minutes  or  less,  then  back  to  saline  until  the  contractions  become  uni- 
form  and   typical  of  the  saline  curve.     Repeat,  varying  the  time  of  the 
immersion  in  the  drug.     Increase  the  strength  of  the  solution  to  0.5  per- 
cent.    Ref. — Lingle;  Am.  Jour.  Physiol.,  VIII,  75. 

3.  Caffeine  on  the  frog's  heart.     Expose  the  heart  of  a  pithed  frog 
and  adjust  a  balanced  lever  on  the  ventricle.     Irrigate  the  surface  of  the 
heart  with  physiological  saline  from  a  dropping  bottle  for  a  few  minutes,  then 
change  the  irrigating  fluid  to  I  percent  caffeine  in  saline  for  five  minutes, 
after  which  return  to  saline  irrigation.     Repeat  once  or  twice,  then  apply 
caffeine  continuously   until   the   maximum   effect  is   obtained.     Compare 
especially  the  auricle  with  the  ventricle  in  the  later  stages  of  the  caffeine 
effect. 

'  When  the  heart  is  perfused  through  one  of  the  veins  then  the  solution 
of  caffeine  should  not  be  stronger  than  o.i  to  0.2  percent,  and  the  record 


CAFFEINE.  21 

should  be  taken  by  the  apex  suspension  method.     This  method  yields  the 
more  accurate  results. 

4.  Caffeine  on  muscular  irritability  and  muscular  work.  Lay 
a  tight  ligature  around  the  thigh  of  a  frog  to  close  off  the  circulation  in  one 
leg.  Give  a  dorsal  lymph  sac  or  abdominal  injection  of  i  c.c.  of  0.5  percent 
caffeine.  Allow  twenty  minutes  for  absorption,  then  pith  the  frog  and 
ligate  off  the  caffeinized  gastrocnemius.  Determine  the  irritability  by 
the  minimal  stimulus  method  and,  second,  determine  the  amount  of 
work  the  muscle  will  do  when  stimulated  directly  once  in  two  seconds 


FIG.  12. — Action  of  caffeine  on  the  ventricular  muscle  of  the  terrapin.  Between 
the  marks  X-X  the  strip  was  immersed  in  0.5  percent  caffeine  in  saline.  Before  and 
after  the  caffeine  the  strip  contracted  in  normal  saline. 

until  completely  fatigued.  Use  a  constant  load  of  50  grams  in  this  experi- 
ment. Prepare  first  the  normal  gastrocnemius  as  quickly  as  possible  and  test 
its  irritability  and  the  amount  of  work  it  will  do  with  uniform  load  and 
method  of  stimulation.  Repeat  the  irritability  test  and  the  determination 
of  work  using  the  caffeinized  muscle.  If  the  records  are  taken  on  the 
same  recording  paper,  one  above  the  other  as  in  experiment  5,  page  3, 
the  comparison  is  very  sharp. 

5.  Caffeine  on  the  voluntary  work  of  human  muscle.  Measure 
the  amount  of  work  of  the  flexors  of  the  middle  finger  by  means  of  a  Mosso's 
ergograph  while  lifting  a  3  to  4  kilo  weight,  one  that  exhausts  the  muscle  in 
about  50  contractions.  Repeat  in  thirty  minutes.  Consider  these  as 
normals.  Take  two  cups  of  strong  coffee  or  0.3  gram  of  caffeine  in 
sweetened  warm  water.  Remeasure  the  work  of  the  flexors  as  directed 


22 


EXPERIMENTAL    PHARMACOLOGY. 


above  60  and  90  minutes  after  taking  the  caffeine.  The  amount  of  the 
muscular  work  is  usually  markedly  increased  by  caffeine.  See  the  next 
experiment. 

6.  Caffeine  on  the  reaction  time  in  man.  Arrange  a  tuning  fork 
vibrating  one  hundred  times  per  second  and  a  set  of  signal  keys  for  measur- 
ing the  reaction  time  to  touch.  (For  details  see  directions  in  Stirling's 
Practical  Physiology,  page  325.)  Determine  the  normal  reaction,  then  the 
reaction  time  at  30,  60,  90  and  120  minutes  after  a  dose  of  0.3  gram 


FIG.  13. 

FIG.  13. — Caffeine  effect  on  the  amount  of  work  of  the  gastrocnemius  of  the  frog. 
The  upper  tracing  is  the  normal,  the  lower  the  opposite  muscle  after  the  absorption  of 

caffeine  in  water.     This  experiment  may  be  performed  together  with  the 
preceding. 

7.  Caffeine  on  the  reflex  time  in  a  frog.  Prepare  a  reflex  frog 
by  destroying  the  brain  and  the  medulla.  After  the  shock  has  passed  away 
determine  the  reflex  reaction  time  to  electrical  stimuli  applied  to  the  toe  of 
the  suspended  frog.  This  time  is  easily  recorded  by  a  paper  writing  point 
attached  to  some  part  of  the  foot  itself.  Give  a  dose  of  I  c.c.  of  0.5  percent 
caffeine  in  the  dorsal  lymph  sac.  After  30  minutes  redetermine  the  reflex 
time.  Draw  conclusions  from  averages  here,  as  the  error  of  procedure  is 
high. 


CAFFEINE. 


23 


8.  Caffeine  on  the  mammalian  heart.  Etherize  a  cat,  bleed  it, 
and  dilute  the  blood  with  Locke's  solution  as  in  the  alcohol  experiment  4. 
Prepare  the  apparatus  and  raise  its  temperature  to  36°  C.  Isolate  the  heart 
and  quickly  insert  it  in  the  cardiograph  and  start  the  circulation  of  the 
Locke-blood  solution.  When  the  rhythm  is  regular,  perfuse  with  o .  o  2  to  0.2 
percent  caffeine  in  Locke-blood  solution.  It  is  better  to  begin  with  a 
weaker  perfusion  solution  of  caffeine  and  increase  the  strength  through  two 
or  three  grades,  say  from  o.i  to  0.5  percent. 


FIG.  13  (Continued). 

The  stimulus  was  repeated  at  regular  intervals  once  in  two  seconds  until  exhaustion. 
0.5  c.c.  i  percent  caffeine. 

9.  Caffeine  on  the  circulation  and  respiration  of  the  mammal. 
Give  a  dog  a  hypodermic  of  i  c.c.  of  2  percent  morphine  and  anesthetize 
with  chloroform.  Tracheotomize.  Take  the  arterial  blood-pressure  from 
the  carotid  and  the  respiratory  pressure  from  the  trachea.  Insert  a  canula 
in  the  saphenous  vein  for  intravenous  injections.  Take  records  on  the 
continuous  paper  kymograph.  After  securing  a  normal  record  of  both  the 
blood-pressure  and  the  respiration  give  an  intraveous  injection  of  5  to  10  c.c. 
of  0.5  percent  caffeine  in  physiological  saline.  Give  slowly  from  a  warmed 
buret.  This  experiment  should  begin  with  the  small  doses  and  the  doses  be 
gradually  increased.  See  the  next  experiment  on  diuretic  action. 


24  EXPERIMENTAL    PHARMACOLOGY. 

10.  Caffeine  as  a  diuretic.  Prepare  a  dog  as  in  the  preceding  experi- 
ment. Take  the  blood-pressure  and  the  respiratory  rate.  Insert  a  canula 
for  venous  injections.  Open  the  abdomen  in  the  median  line,  seek  out  one 
ureter  near  its  connection  with  the  bladder,  ligate  and  insert  a  urethral 
canula,  taking  care  to  make  an  unobstructed  connection.  After  the  flow 
of  secretion  has  been  established,  a  2  to  3  mm.  rubber  tube  is  connected  with 
the  canula  and  the  abdominal  opening  sewed  up.  Connect  the  rubber  tube 
with  a  i  c.c.  pipet  graduated  in  o.i  c.c.  Mount  the  buret  horizontally. 
Read  the  rate  of  secretion  by  injecting  a  small  bubble  of  air  each  five  min- 
utes into  the  rubber  tube  at  its  connection  with  the  buret. 

Determine  the  rate  of  secretion  under  constant  anesthesia  both  before 
and  after  5  to  10  c.c.  of  0.5  percent  caffeine.  Take  readings  every  five 
minutes  for  a  period  of  about  two  hours. 

STRYCHNINE. 

List  of  Experiments  Showing  the  Effects  of  Strychnine.         PAGE. 

1.  On  the  frog 24 

2.  On  a  mammal,  demonstration 24 

3.  On  the  ventricular  strip   . 25 

4.  On  the  frog's  heart  and  cardiac  nerves 25 

5.  On  the  irritability  and  work  of  voluntary  muscle    ....  26 

6.  On  reflex  irritability  and  reaction  time.    . 26 

7.  Local  action  on  the  spinal  cord      26 

8.  Spasms  depend  upon  cutaneous  stimulation 27 

9.  Absorbed  slowly  from  the  stomach  or  bladder  and  readily 

from  the  intestine  or  peritoneum 27 

10.  Stored  in  the  spinal  cord 27 

11.  On  the  mammalian  heart 28 

12.  On  the  blood-pressure  and  respiration  rate  of  mammals.  28 

1.  Strychnine  on  the  frog.     Give  a  frog  a  toxic  dose  of  strychnine 
nitrate,  0.3  c.c.  (5  minims)  of  o.i  percent.     Give  in  the  dorsal  lymph  sac. 
Note  the  time  of  the  appearance  and  the  successive  stages  of  increased 
irritability,  convulsions,  and  paralysis.     Take  fresh  frogs  and  determine  the 
limits  of  the  therapeutic  or  non- toxic  dose,  i.e.,  determine  the  dose  per  gram 
of  body  weight  that  will  just  fail  to  produce  convulsions. 

2.  Strychnine  on  the  mammal.     Demonstration.     Give  a  rabbit  a 
hypodermic  injection  of  0.4  c.c.  of  o.i  percent  strychnine  nitrate  per 


STRYCHNINE.  25 

kilo  of  body  weight.  Consult  Cushny's  Pharmacology  for  symptoms. 
Meltzer  gives  the  toxic  dose  for  rabbits  as  0.5  mgr.  per  kilo.  Reference, 
Am.  Jour.  Physiol.,  Volume  IX,  page  i. 

3.  Strychnine  on  the  ventricular  strip.  Suspend  a  strip  of  terrapin's 
ventricle  in  physiological  saline  and,  when  it  is  contracting  regularly,  subject 
it  to  a  bath  of  o.i  percent  strychnine  nitrate  in  saline  for  five  minutes. 
Return  the  strip  to  physiological  saline  until  the  contractions  are  unques- 
tionably of  the  saline  type.  The  rhythm  and  amplitude  are  both  reduced. 


FIG.  14. — Action  of  strychnine  solution  on  the  ventricular  muscle  of  the  terrapin 
In  trace  a  the  strip  was  contracting  in  physiological  saline.  At  the  vertical  arrow  it 
was  transferred  to  0.5  percent  strychnine.  The  contractions  are  gradually  slowed  and 
weakened.  Tracings  b  to  g  are  successive  records  around  a  six-inch  drum  during  the 
recovery  which  is  very  prolonged  in  this  experiment. 

4.  Strychnine  on  the  heart  and  cardiac  nerves.  Pith  a  frog,  expose 
the  heart,  take  a  direct  tracing  of  the  movements  of  the  ventricle.  Test 
the  vagus  activity  by  stimulating  the  vago-sympathetic  trunk  with  a  current 
that  produces  complete  inhibition.  Next  irrigate  the  surface  of  the  heart 
from  a  dropping  bottle  with  o.i  percent  strychnine  nitrate  in  physiological 
saline.  While  continuing  the  irrigation  stimulate  the  vagus  trunk  at  inter- 
vals of  5  to  10  minutes.  Look  for  a  progressive  effect  on  the  beat,  the  rate, 
and  on  the  cardiac  nervous  mechanism  as  demonstrated  by  the  results  of  the 
stimulation. 


26  EXPERIMENTAL  PHARMACOLOGY. 

5.  Strychnine  on  the  irritability  and  work  of  voluntary  muscle. 

Ligate  the  thigh  of  one  leg  of  a  frog  to  occlude  its  circulation,  give  a  dose 
of  0.2  c.c.  of  o.i  percent  strychnine  nitrate.  In  twenty  minutes  ligate 
off  the  other  leg,  pith  the  frog  (pith  immediately  if  tetanic  contractions 
appear  earlier),  and  determine  the  irritability  of  the  normal  and  of  the 
drugged  gastrocnemii  by  the  minimal  stimulus  method. 

Follow  the  above  test  by  measurement  of  the  work  the  muscles  will 
do,  testing  first  the  normal,  and  second,  the  strychnine  muscle.  Use  the 
method  described  for  experiment  5  under  alcohol.  The  irritability  and 
the  work  of  voluntary  muscle  are  both  greatly  increased  in  the  therapeutic 
stage,  in  sharp  contrast  to  the  effect  of  strychnine  on  heart  muscle. 


IIIIIHIHIIIIIIII...I 


FIG.  15 — Showing  the  action  of  strychnine  on  the  muscular  work  and  on  amplitude 
of  the  single  contractions  in  the  frog's  gastrocnemius.  The  lower  series  is  of  the  normal 
muscle,  the  upper  of  the  strychninized  muscle.  The  dose  was  0.25  c.c.  of  o.i  percent 
strychnine  hydrochloride  injected  into  the  dorsal  lymph  sac,  after  %ation  of  one  leg  at 
the  thigh.  Absorption  was  allowed  until  the  first  spasms  when  the  frog  was  pithed. 
Weight  of  frog  40  grams.  Load  200  grams.  One  stimulus  in  three  seconds. 

6.  Strychnine  on  reflex  irritability  and  reaction  time.    Prepare  a 
reflex  frog,  i.  e.,  destroy  only  the  brain  including  the  medulla.     After  the 
shock  has  passed  away,  one  hour  or  more,  determine  the  reflex  reaction  time 
to  electrical  stimulation  of  the  toe  by  the  method  given  on  page  71.     Now 
give  0.5  c.c.  (8  minims)  of  0.02  percent  strychnine  nitrate.     After  each 
ten  minutes  take  the  reaction  time  to  electrical  stimulation  until  spasms 
appear,  which  ought  to  be  under  60  minutes. 

7.  Local  action  of  strychnine  on  the  spinal  cord.     Cut  the  cord  of  a 
frog  at  the  base  of  the  medulla  and  destroy  the  brain.     Free  the  cut  end  of 
the  cord  from  the  surrounding  tissue,  and  carefully  paint  it  with  I  percent 
strychnine  nitrate  solution.     Muscular  spasms  will  be  produced  or  will 
follow  stimulation  of  the  toes  of  the  fore  leg.     Or  stimulate  the  toe  of  the 


STRYCHNINE.  27 

hind  leg — reflexes  of  an  orderly  nature  occur,  where  no  general  tetanic  con- 
vulsions have  been  induced  by  the  dose.     Pith  the  cord,  all  spasms  cease. 

8.  Strychnine  spasms  depend  also  upon  cutaneous  stimulation. 
Strychninize  a  frog  and  when  the  spasms  are  strong  and  continuous  paint 
the  skin  with  a  2  percent  cocaine  solution.     The  cocaine  paralyzes  the 
cutaneous  sensory  apparatus  whereupon  the  convulsions  cease.     Dip  the 
frog  in  water  to  remove  the  excess  of  cocaine,  its  local  effect  will  disappear 
in  about  10  minutes  and  the  strychnine  convulsions  will  reappear. 

9.  Strychnine  is  absorbed  very  slowly  from  the  stomach  or  blad- 
der, but  very  readily  from  the  intestine  and  body  cavity.    Anesthetize 
a  half-grown  fasting  cat.     Ligate  both  the  cardiac  and  the  pyloric  orifices 


FIG.  16. — Strichnine  on  the  isolated  mammalian  heart.  The  heart  was  perfused 
with  Locke's  solution  containing  10  percent  of  the  animal's  own  blood.  Between  the 
points  marked  on  and  off,  a  total  of  50  seconds,  the  heart  was  profused  with  o.oi  percent 
strychnine  hydrochloride.  The  stock  solution  of  the  drug  had  been  carefully  neutralized 
to  eliminate  any  trace  of  acid  effects.  Temperature  34°  C.  Time  in  seconds. 

of  the  stomach.  Inject  10  c.c.  of  o.i  percent  (10  mgrs.)  of  strychnine 
nitrate  into  the  stomach.  If  no  spasms  occur  in  30  minutes  then  cut  the 
pyloric  ligature  and  run  the  stomach  content  into  the  intestine.  Spasms 
may  be  looked  for  in  two  minutes  or  less.  If  enough  strychnine  is  absorbed 
from  the  stomach  to  produce  muscular  spasms  repeat  the  second  half  of  the 
experiment  on  a  second  animal,  injecting  the  drug  directly  into  a  portion  of  the 
intestine. 

Compare  absorption  from  the  urinary  bladder  and  from  the  abdominal 
cavity  in  the  same  manner. 

10.  Strychnine  is  stored  in  the  spinal  cord  (Lovett,  Jour.  PhysioL, 
Volume  I,  p.  99).  Inject  10  mgs.  strychnine  nitrate  into  the  dorsal 
lymph  sac  of  a  large  bull  frog.  Allow  30  minutes  for  absorption.  Then 
remove  the  skin  and  wash  away  all  traces  of  strychnine  that  may  re- 


26  EXPERIMENTAL    PHARMACOLOGY. 

•main  unabsorbed.  Take  the  cord,  also  an  equal  amount  of  other  tissue, 
macerate  each  in  0.7  percent  saline.  Inject  equal  portions  of  the  extracts  in 
the  dorsal  lymph  sacs  of  two  frogs.  Allow  hours,  if  necessary,  for  the  symp- 
toms to  develop.  This  method  will  detect  traces  of  strychnine  too  small  for 
chemical  identification. 

11.  Strychnine  on  the  mammalian  heart.     Arrange  the  apparatus 
for  the  isolated  mammalian  heart,  bring  it  to  a  temperature  of  34°  to  36°  C. 
Anesthetize  a  cat  or  rabbit  with  ether,  bleed  and  defibrinate  the  blood  and 
dilute  it  to  ten  volumes  with  Locke's  solution.     Quickly  cut  out  the  heart, 
insert  the  canula  into  the  aorta  and  ligature  it,  attach  the  cardiograph,  and 
start  the  perfusion  of  Locke-blood  solution.     Perfuse  with  0.005  percent 
strychnine  hydrochloride  for  30  secods.     The  dose  may  be  increased  to 
o.o i  or  even  0.02  percent  but  recovery  from  the  latter  is  very  slow  and 
gradual. 

12.  Strychnine  on  the  blood-pressure  and  on  the  respiration  rate 
of  mammals.     Anesthetize  a  lo-kilo  dog.     Take  a  continuous  record  of  the 
blood-pressure  from  the  carotid  and  of  the  respiration  rate  from  the  trachea. 
Give  an  injection  of  i  c.c.  of  o.i  percent  solution  of  strychnine  nitrate 
from  a  hypodermic  into  the  saphenous  vein.     One  should  give  close  atten- 
tion to  the  symptoms  of  this  mild  dose  which  will  produce  little  more  than 
the  therapeutic  effects.     Repeat  this  injection  until  the  cumulative  dose  pro- 
duces convulsions  of  a  mild  character.     Note  that  the  convulsions  may  be 
suppressed  here  by  giving  more  chloroform.     Give  especial  attention  to  the 
blood-pressure  conditions  during  the  tetanic  spasm,  so  as  to  eliminate  the 
strictly  passive  mechanical  factors. 


COCAINE. 


COCAINE. 

List  of  Experiments  Showing  the  Action  of  Cocaine.  PAGE. 

1.  On  the  frog 29 

2.  On  local  sensory  surfaces 29 

3.  On  the  heart  muscle 30 

4.  On  the  frog's  heart 30 

5.  On  muscle  work 30 

6.  On  the  circulatory  and  respiratory  systems  of  the  mammal .  31 

1.  Cocaine  on  the  frog.     Give  a  frog  a  dorsal  lymph  sac  injection 
of  0.4  c.c.  of  0.5  percent  solution  of  cocaine  hydrochlorate  in  physio- 
logical saline.     Observe  the  symptoms  in  the  usual  way.     Also  examine 
the  white  corpuscles  of  the  blood  for  motility  as  compared  with  the  unpoisoned 
frog. 

2.  Cocaine  on  local  sensory  surfaces.     Paint  one-half  the  surface 
of  your  own  tongue  with  a  brush  wet  in  2  percent  solution  of  cocaine 


FIG.   17. — Cocaine  effects  on  the  frog's  heart  when  perfused    in    o.ooi    percent 
Ringer's   solution.     Time    in    seconds. 

hydro  chloride.  Use  care  not  to  swallow  any  of  the  solution.  In  8  to  10 
minutes  compare  the  sensitiveness  of  the  two  halves  of  the  tongue  to  electri- 
cal currents  by  the  minimal  stimulus  method.  Test  for  taste  sensations  of 
sweet,  of  salt.  Note  also  the  personal  sensations  of  any  character  result- 
ing from  the  experiment. 

Give  one  drop  of  i  percent   cocaine  in  the  right  eye;  repeat  in 
three  minutes.     There  is  a  loss  of  sensitiveness  and  the  eyeball  may  be 


30  EXPERIMENTAL    PHARMACOLOGY. 

touched  without  pain.  Compare  the  pupils  as  to  size,  as  to  reaction  to 
light.  Determine  the  acuteness  of  vision  of  each  eye  separately  at  the  read- 
ing distance.  Cocaine  is  an  analgesic,  but  not  a  perfect  mydriatic. 

3.  Cocaine  on  the  heart  muscle.  Prepare  a  strip  of  terrapin's 
ventricle  and  when  it  is  beating  in  physiological  saline  in  good  rhythm 
submit  it  for  from  three  to  five  minutes  to  a  bath  of  o.oi  percent  cocaine  in 
saline.  Record  the  contractions  on  a  slow  drum. 


FIG.  18. 
FIG.  18. — Effect  of  cocaine  on  the  muscular  work  of  the  frog's  gastrocnemius. 

4.  Cocaine  on  the  frog's  heart  and  its  nervous  mechanism.    Pith 
a  frog,  expose  the  heart.     Take  a  continuous  record  of  its  contractions  by 
the  usual  method.     Irrigate  its  surface  with  physiological  saline.     Test  the 
irritability  of  the  vagus  trunk  using  a  medium  strength  stimulus.     Now 
irrigate  the  heart  for  one  minute  with  0.2  percent  cocaine  in  saline  followed 
by  saline.     When  the  cocaine  contractions  have  somewhat  recovered,  retest 
the  inhibitory  power  of  the  vagus.     Repeat  the   test,using   two  or  three 
drops  of  i  percent  solution,  and  do  not  wash  it  off  afterward. 

Perfuse  the  heart  with  0.002  percent  solution  of  cocaine  in  Ringer's 
weaker  solution.  This  method  gives  more  constant  results  than  does  the 
irrigation  and  it  is  to  be  preferred. 

5.  Cocaine  on  muscle  work.     Ligate  one  leg  of  a  frog  at  the  thigh,  or 
use  the  method  described  under  alcohol.     Give  a  dose  of  0.4  c.c.  (7  minims) 
of  0.5  percent  cocaine  in  the  dorsal  lymph  sac.     Allow  20  minutes  for 
absorption  then  ligate  the  cocainized  leg.     Load  with  a  5o-grain  weight. 


COCAINE.  31 

Take  records  of  the  contractions  of  the  normal  muscle  on  a  drum  with  a 
speed  of  i  mm.  per  second.  Stimulate  with  single  break  induction  shocks 
once  in  two  seconds  until  exhausted.  Prepare  the  cocainized  gastrocnemius, 
mount  and  stimulate  with  the  same  rate  and  load.  If  the  two  records  are 
parallel  on  the  same  paper,  see  figure  18,  it  will  demonstrate  the  comparative 
difference  in  work  done.  Calculate^the  amount  of  work  per  gram  of  muscle 
in  each  of  the  two  preparations. 


FIG.  18  (Continued). 
The  endurance  of  the  cocainized  muscle  is  greatly  increased. 

6.  Cocaine  on  the  circulatory  and  respiratory  systems  of  the 
mammal.  Give  morphine  and  chloroform  to  a  dog.  Insert  a  tracheal 
canula.  Take  the  blood-pressure  and  respiratory  records  on  the  continuous 
paper  kymograph.  Insert  a  canula  and  connect  a  buret  with  the  saphenous 
vein.  Inject  2  c.c.  of  i  percent  cocaine  very  slowly  while  watching 
the  blood-pressure  as  an  indicator. 


32  EXPERIMENTAL    PHARMACOLOGY. 

QUININE. 

Experiments  on  the  Effect  of  Quinine.  PAGE. 

1.  On  the  frog.   .    .    .    v   ...,....,,..,,    ,-.;.  ;      32 

2.  On  the  frog's  heart.      v-  .    .    .    ...    .    .  -,,,.,    ......  .     32 

3.  On  the  striated  muscle     .'  .    .    .    .    .    .    ,  -.  >  ,..--.    .    .  .     32 

1.  Quinine  on  the  frog.     Inject  i  c.c.  of  o.i  percent  solution  of 
hydrochlorate  of  quinine  into  the  dorsal  lymph  sac.     In  addition  to  the 
usual  observations,  examine  the  blood  of  this  frog  as  regards  the  motility 
of  the  white  corpuscles.     Compare  with  the  blood  of  a  normal  frog. 

2.  Quinine  on  the  frog's  heart.     Pith  a  frog,  expose  the  heart,  and 
take  a  record  of  its  contractions  with  physiological  saline  while  perfusing 
it  from  a  perfusion  bottle.     Change  the  Ringer's  fluid  to  0.05  percent 
quinine  hydrochlorate  in  Ringer  for  about  one  minute.     Repeat  after 


WWW 


0.05%  Quinine    R 


FIG.  19. — Action  of  quinine  on  the  frog's  heart.  The  perfusion  method  was  used. 
Normal  rate  with  Ringer's  solution.  Strength  of  quinine  0.05  per  cent.  Time  in 
half  minutes. 

recovery,  using  I  percent  quinine.  Continue  this  perfusion  until  no  further 
contractions  are  secured.  Examine  the  condition  of  the  ventricle  at  the  close 
of  the  experiment. 

Vary  this  experiment  by  pithing  the  frog,  taking  care  to  lose  little  blgod 
and  making  a  record  from  the  ventricle.  Now  give  a  lymph  sac  injection 
of  i  c.c.  of  i  percent  quinine  and  take  a  continuous  record  through  20  to 
30  minutes.  | 

3.  Quinine  on  the  striated  muscle.  Ligate  one  leg  of  a  frog  at 
the  thigh,  inject  i  c.c.  of  o.i  percent  quinine  into  the  dorsal  lymph  sac. 
In  just  20  minutes  ligate  off  the  other  leg  and  pith  the  frog.  Determine 
the  irritability,  first  of  the  normal  then  of  the  drugged  muscle.  Determine 
the  work  each  gastrocnemius  will  do. 


ATROPINE. 


33 


ATROPINE. 

Experiments  Showing  the  Action  of  Atropine.  PAGE. 

i.  On  the  frog 33 


2.  On  the  heart  muscle 

3.  On  the  frog's  heart  and  on  the  cardiac  nervous  apparatus 

4.  On  the  secretory  nerves  of  a  mammal 

5.  On  the  isolated  heart  of  the  cat 

6.  On  the  circulatory  and  respiratory  systems  of  the  mammal 

7.  On  the  eye      

8.  On  man  in  the  therapeutic  dose 


33 
34 
34 
35 
35 
36 
36 


9.  As  secreted  by  the  kidney    ...............     36 

10.  Scopolamine  on  the  frog 36 

i.  Atropine  on  the  frog.  Give  a  frog  an  injection  of  i  c.c.  of  i 
percent  of  atropine  sulphate.  Keep  it  under  observation  in  a  moist 
battery  jar  until  complete  recovery.  The  toxic  dose  is  i  c.c.  of  3  percent. 


Atropine 


FIG.  20. — Action  of  atropine  on  the  heart  muscle  of  the  terrapin's  ventricle. 
The  normal  solution  is  sodium  chloride,  the  strength  of  atropine  o.ooi  percent.  The 
decrease  in  amplitude  is  usually  not  so  great  as  in  this  tracing,  or  is  even  absent  entirely 
Time  in  seconds. 

2.  Atropine  on  the  heart  muscle.  Mount  a  ventricular  strip  from 
the  terrapin  in  0.7  percent  sodium  chloride,  and  when  it  is  contracting  with  a 
uniform  amplitude  and  regular  rhythm  change  to  o.ooi  percent  atropine 
in  physiological  saline  for  five  minutes,  see  figure  17.  Recover  the  charac- 
teristic rhythm  in  saline  and  repeat  using  0.002  percent  atropine  in  saline.. 

3 


34 


EXPERIMENTAL  PHARMACOLOGY. 


3.  Atropine  on  the  frog's  heart  and  cardiac  nervous  apparatus. 

Pith  a  frog,  expose  the  heart  and  take  a  tracing.  Determine  an  effective 
strength  of  stimulus  for  the  vagus.  Now  irrigate  the  heart  for  one  minute 
from  a  dropping  bottle  containing  o.i  percent  atropine  in  saline.  Stimu- 
late the  vagus  immediately  and  once  every  five  minutes  or  less.  Atropine 
eliminates  the  vagus  control  of  the  heart  by  poisoning  the  peripheral  endings. 
The  atropine  effect  is  antagonized  by  physostigmine,  page  44,  experiment  3, 
and  by  muscarine.  , 

4.  Atropine  on  the  secretory  nerves  of  a  mammal.    Anesthetize 
a  10  kilo  dog  with  morphine  and  chloroform.     Expose  and  tie  a  canula  in 


FIG.  21. 

'•  FIG.  2 1.  —Action  of  atropine  on  the  isolated  heart  of  the  cat.  Locke-blood  solution 
the  arrows  was  .001  percent.  Temperature  39°  C.  Pressure  85  cm.  of  water.  The 

the  subrhaxillary  duct.  Expose  and  stimulate  the  chorda  tympani  nerve 
in  the  hilus  of  the  gland,  noting  the  rate  of  secretion  by  the  drops  of  saliva 
per  minute  from  the  canula.  Give  a  hypodermic  injection  of  1.5  c.c.  of 
i  percent  atropine.  Stimulate  the  chorda  tympani  nerve  again.  No 
secretion  is  obtained  even  though  the  nerve  is  stimulated  down  close  to  the 
hilus  of  the  gland. 

,  This  demonstration  may  be  made  in  part  as  follows :  Produce  a  rapid 
flow  of  sajvva  in  the  dog  by  a  hypodermic  of  i  c.c.  of  0.2  percent  pilocarpine. 
Observe,  the  flow  by  turning  out  the  dog's  upper  lip.  Follow  with  a  hypo- 
dermic fjose  of  i  c.c.  of  i  percent  atropine.  The  secretion  stops. 


ATROPINE. 


35 


5.  Atropine  on  the  isolated  heart  of  the  cat.    Anesthetize  a  cat 
with  ether.     Bleed  it  and  prepare  the  Locke-blood  perfusion  fluid.     Isolate 
and  suspend  the  heart  in  the  perfusion  apparatus.     Obtain  a  normal  record 
then  perfuse  with  .001  percent  atropine  in  Locke-blood  solution. 

6.  Atropine  on  the  circulatory  and  respiratory  systems  in  the 
mammal.     Anesthetize  a  lo-kilo  dog  as  in  experiment  4  preceding.     Place 
an  arterial  canula  in  the  carotid  and  insert  a  tracheal  canula.     Take  a  con- 
tinuous record  of  the  blood-pressure  and  of  the  respiration.     Stimulate  the 
peripheral  end  of  the  sectioned  vagus  with  a  stimulus  that  produces  complete 
inhibition  of  the  heart.     Stimulate  also  the  central  end  of  the  vagus.     Now 


FIG.  21  (Continued). 

The  strength  of  atropine  perfused  between 


was  used  for  the  normal  perfusion  fluid, 
time  in  seconds. 


give  an  intravenous  injection  of  i  c.c.  of  o.i  percent  atropine.  Note 
the  exact  time  of  the  injection  on  the  record  by  a  signal  pen.  When  the 
equilibrium  is  again  established,  re-stimulate  the  ends  of  the  sectioned  vagus 
with  the  same  strengths  of  stimulus  used  before  atropine  was  given.  Note 
that  the  heart  rate  is  no  longer  slowed  on  stimulation  of  the  peripheral 
vagus,  but  that  the  pupil  still  actively  dilates  after  this  dose  when  the 
central  end  of  the  vagus  trunk  is  stimulated.  Atropine  also  destroys  the 
vagus  control  over  the  smooth  muscle  of  the  alimentary  tract  thus  decreasing 
its  motility. 

Physostigmine  is  an  antagonist  to  atropine.     Try  i  c.c.  of  o.i  percent 


36  EXPERIMENTAL    PHARMACOLOGY. 

intravenous.     Use  artificial  respiration  if  necessary.     Give  a  second  injec- 
tion of  atropine  later.     See  figure  23  for  a  reverse  antagonism. 

7.  Atropine  on  the  eye.     Drop  i  or  2  drops  of  i  percent  atropine 
in  the  right  eye  of  a  dog  or  cat.     The  pupil  will  be  widely  dilated  in  a  few 
minutes.     Keep  the  animal  under  observation  until  the  effect  entirely  dis- 
appears, often  only  after  several  days.     Atropine  destroys  the  power  of 
accommodation  and  it  is  used  for  this  clinical  purpose  in  eye  practice. 
Students  should  not  use  atropine  on  their  own  eyes,  but  a  mild  dose  of  hom- 
atropine,  2  or  3  drops  of  i  percent,  the  effect  of  which  passes  off  in  24 
to  36  hours,  may  be  tested  in  one's  own  eye.     In  such  experiments  test  the 
accommodation,  light  reflex,  and  size  of  the  pupil. 

8.  Atropine  on  man  in  therapeutic  dose.    Test  on  yourself  the 
action  of  a  dose  of  1/120  to  1/60  grain  of  atropine  by  way  of  the  mouth. 
Note  the  effects  on  the  heart  rate,  pulse  character,  respiration,  size  of  pupil, 
light  reflex  and  sensations. 

9.  Atropine  is  secreted  by  the  kidney. — This  may  be  demonstrated 
on  the  rabbit  which  is  very  tolerant  of  the  drug.     Give  a  rabbit  urethane. 
Collect  the  urine  from  a  bladder  canula.     Give  a  large  hypodermic  injection, 
2  c.c.  of  2  percent  atropine,  and  test  the  rabbit's  urine  on  the  eye  of  a  cat  or 
dog.     The  atropine  may  be  extracted  (Binz).     Concentrate  a  large  amount 
of  urine,  add  ammonia,  shake  up  with  chloroform,  evaporate,  dissolve  the 
residue  and  test  on  the  eye  of  a  cat  or  a  dog. 

10.  Scopolamine  on  the  frog.     Give  a  dose  of  i  c.c.  of  i  percent 
scopolamine  in  the  dorsal  lymph  sac  of  a  frog.     Compare  with  the  effects 
of  an  equal  dose  of  atropine  in  experiment  i  above. 


NICOTINE.  37 


NICOTINE. 

Experiments  Illustrating  the  Action  of  the  Nicotine.  PAGE. 

1.  On  the  frog 37 

2.  On  the  ventricular  muscle 37 

3.  On  the  frog's  heart  and  its  nervous  apparatus 37 

4.  On  the  nerve  fiber  and  on  nerve  ganglia 37 

5.  On  the  mammalian  heart 38 

6.  On  the  circulatory  system  and  on  the  respiratory  nervous 

mechanism   .    .    .    .    .    ,   . .    .    .    .    .    ....     38 

7.  On  muscle  irritability •  .    .    .     39 

1.  Nicotine  on  the  frog.     Give  an  injection  of  a  0.5  c.c.  of  0.2 
percent  nicotine  into  the  dorsal  lymph  sac  of  a  frog. 

2.  Nicotine  on  the  ventricular  muscle.     Prepare  a  terrapin's  heart 
strip  and  when  it  is  contracting  rhythmically  in  0.7  percent  physiological 
saline,  immerse  the  strip  in  a  0.05  percent  solution  of  nicotine  in  saline 
for  two  minutes,  then  return  to  the  saline  bath.  "  Repeat.     If  the  solution  is 
too  strong  the  strip  will  exhibit  a  strong  tonus  with  incomplete  relaxations. 
The  amplitude  and  the  rate  are  markedly  increased. 

3.  Nicotine   on   the   frog's  heart  and  its  nervous  mechanism. 
Pith  a  frog,  expose  the  heart  and  take  tracings  on  a  drum  with  a  speed  of 
2   mm.  per  second.     Stimulate  the  vago-sympathetic  with  an  interrupted 
current  that  just  causes  complete  inhibitions.     Now  irrigate  the  heart  from 
a  dropping  bottle  with  o.i  percent  nicotine  in  0.7  percent  saline,  and 
stimulate  the  vagus  at  intervals  of  two  minutes.     If  the  nerve  stimulation 
ceases  to  be  effective,  then  apply  the  electrodes  directly  to  the  sinus. 

To  demonstrate  the  stronger  effects  on  heart  muscle  prepare  a  second 
frog.  Take  a  tracing  of  the  heart.  Apply  a  few  drops  of  i  percent  solu- 
tion of  nicotine. 

4.  Nicotine  on  the  nerve  ganglia  and  on  the  nerve  fiber.  Anes- 
thetize a  rabbit  (or  cat  or  dog),  dissect  out  the  cervical  sympathetic  and 
the  superior  cervical  ganglion.     Stimulations  of  the  nerve  or  of  the  ganglion 
lead  to  vasoconstriction  in  the  ear  and  dilation  of  the  pupil.     Paint  the 
nerve  below  the  ganglion  with  i  percent  nicotine.     Stimulation  at  a  point 
still   lower  down  shows  that   the   nerve  impulses  still   pass  undisturbed. 
Now  paint  the  ganglion  itself.     Stimulate  the  nerve  below  the  ganglion,  also 


38  EXPERIMENTAL    PHARMACOLOGY. 

the  ganglion  directly.  What  Conclusions  ?  See  also  the  next  experiment. 
(Ref. :  Langley  and  Dickinson,  Journal  of  PhysioL,  Volume  II,  page  265.) 
5.  Nicotine  on  the  mammalian  heart.  Prepare  the  mammalian 
heart  perfusion  and  recording  apparatus  and  bring  it  to  a  temperature  of 
36°  C.  Etherize  a  cat,  bleed,  defibrinate  the  blood,  and  dilute  to  one-in-ten 
of  Locke's  solution.  Quickly  take  out  the  heart,  suspend  it  in  the  apparatus 
and  start  the  perfusion.  When  the  heart  is  beating  well,  perfuse  it  with 
o.ooi  percent  of  nicotine  in  Locke-blood  solution.  There  is  a  constant 


FIG.  22. — Action  of  nicotine  on  the  ventricular  muscle  from  the  terrapin.  The 
strip  was  contracting  in  physiological  saline  when  transferred  to  0.2  percent  nicotine  in 
saline  at  the  point  marked.  The  solution  was  too  strong  and  was  removed  after  about 
forty  seconds.  Time  in  seconds  and  half-minutes. 


sharp  increase  in  amplitude  with  a  more  slowly  developed  increase  in 
rate.  The  after  amplitude  may  remain  greater  than  the  preceding  normal. 
6.  Nicotine  on  the  circulatory  and  respiratory  nervous  mechan- 
ism. Anesthetize  a  dog  or  cat  (the  animal  used  in  experiment  4  above 
may  be  used  for  this  experiment  also).  Take  a  blood -pressure  from  the 
carotid  and  a  respiration  tracing  from  the  trachea,  (i)  Determine  an  effect- 
ive stimulus  for  the  heart  and  respiration.  (2)  Now  inject  5  c.c.  of  o.i 
percent  solution  of  nicotine  into  the  saphenous  or  jugular  vein.  Repeat 


NICOTINE. 


39 


the  dose  if  necessary,  until  distinct  effects  are  produced  on  the  heart  rate  and 
blood-pressure.  (3)  Stimulate  the  vagus  at  first  with  the  strength  of  stimu- 
lus used  before  the  injection,  then  with 'successively  stronger  stimuli.  An 
instructive  picture  is  obtained  by  dissecting  down  to  and  stimulating  the 
cardiac  branches  from  the  annulus  of  Vieussens,  which  may  be  done  in 
the  dog  on  the  left  side  without  opening  the  thorax. 


FIG.  23. — Action  of  nicotine  on  the  isolated  cat's  heart.  The  heart  was  contract- 
ing in  Locke-blood  solution  when  it  was  perfused  with  o.ooi  percent  nicotine  between 
the  points  marked  by  the  arrows.  The  increase  in  amplitude  is  more  marked  when 
the  heart  is  beating  weaker  at  the  time  of  perfusion.  Time  in  seconds. 

7.  Nicotine    on  muscle    irritability.     Lay   a   ligature   around   the 
thigh  of  one  leg  of  a  frog  and  then  give  i  c.c.  of  o.i  percent  nicotine 

in  the  dorsal  lymph  sac.  After  20  minutes  test  the  irritability  of  the  normal 
and  of  the  nicotinized  gastrocnemius  muscles  by  the  minimal  and  maximal 
stimulus  method. 


40  EXPERIMENTAL    PHARMACOLOGY. 


CURARE. 

Experiments  on  the  Effect  of  Curare.  PAGE. 

1.  On  the  frog.    .    .    ^ ;  y:  ......  ...-,..- ','-.;  v  '•$•';,* .  ,  \  r.  ....  40 

2.  On  the  motor  nerve  endings.    .    .    .    .    .    .    .    .  f.    .    ...  40 

3.  On  the  heart  muscle  and  on  the  cardiac  nervous  mechan-  , 

ism.   .v '• .  -..-..  ••:•"-.-  .":"v>sv~",  •'.'•."'.. -"  .  '.    . 40 

4.  Poisons  the  motor  endings  before  the  other  portions  of  the 

reflex  arc.     .    .    .    ,  ...    ...  ...    .    .    . 41 

5.  On  the  mammal.   .    .    .    .   ;.    ,    .-   .    ,    ...    ...  .    ....  41 

i.  Curare  on  the  frog.  Give  a  frog  a  hypodermic  of  0.3  c.c.  (5 
minims)  of  0.2  percent  curare.  The  motor  apparatus  is  paralyzed,  but 
the  circulation  continues  and  the  frog  will  recover  in  from  one  to  three  days, 
respiration  being  maintained  through  the  moist  skin  if  the  animal  is  kept  in 
a  covered  jar. 

.2.  Curare  on  the  motor  nerve  endings,  Bernard's  experiment. 
The  specific  action  of  curare  was  demonstrated  by  Claude  Bernard  to  be  on 
the  motor  nerve  end  plates.  Ligate  one  leg  of  a  frog  to  shut  off  the  circula- 
tion, give  a  hypodermic  of  0.3  c.c.  of  0.2  percent  curare.  When  general 
paralysis  is  secured,  perform  the  following  tests,  interpreting  the  results 
through  the  effect  on  the  gastrocnemius  muscles: 

a.  Stimulate  the  sciatic  nerve  on  the  unligated  leg. 

b.  Stimulate  the  gastrocnemius  of  the  unligated  side. 

c.  Stimulate  the  sciatic  nerve  on  the  ligated  side  above  the  ligature. 

d.  Below  the  ligature. 

e.  The  corresponding  muscle.     Conclusions. 

3.  Curare  on  the  heart  muscle  and  on  the  cardiac  nervous  appa- 
ratus. While  minimal  doses  of  curare  suffice  to  poison  the  motor  end  plates, 
it  takes  relatively  large  doses  to  paralyze  the  cardiac  nervous  apparatus. 
The  paralysis  apparently  affects  the  ganglionic  nerve  endings  first  and  then 
the  cardiac  motor  endings  and  muscle.  Pith  a  frog,  expose  the  heart,  pre- 
pare the  vagus  trunk  for  stimulation  and  adjust  a  heart  lever  for  record. 
Allow  physiological  saline  from  an  irrigating  bottle  to  run  over  the  heart. 
Take  a  normal  record  and  then  stimulate  the  vagus  nerve.  Now  irrigate 
slowly  with  0.2  percent  curare  in  saline  and  stimulate  the  nerve  10  seconds 
at  a  time  at  intervals  of  10  minutes  for  several  tests. 


CURARE.  41 

4.  Curare  poisons  the  motor  endings  before  the  other  portions 
of  the  reflex  arc.     Tie  a  ligature  on  a  frog's  leg  at  the  thigh,  inject  0.3 
c.c.  of  0.2  percent  curare.     Just  as  voluntary  activity  ceases  stimulate  the 
skin  of  the  poisoned  leg.     The  unpoisoned  gastrocnemius  will  contract. 
Rapidly  expose  and  stimulate  the  poisoned  sciatic.     The  poisoned  gastroc- 
nemius will   not  contract,  while  the  unpoisoned  one  will,  owing  to  reflex 
stimulation  through  the  cord. 

5.  Curare   on   the  mammal.     Morphinize  and  chloroform  a  dog. 
Take  blood-pressure.     Introduce  a  tracheal  canula  and  take  the  respiratory 
record  by  the  intra- tracheal  method.     Arrange  the  apparatus  for  artificial 
respiration  when  needed.     Inject  into  a  vein  5  c.c.  of  i  percent  curare. 
All  movements  of  voluntary  muscles  will  quickly  cease  including  respiratory 
movements.     The  heart  rate  and  the  blood-pressure  remain  good,  and  if 
artificial  respiration  is  applied  the  circulation  can  be  maintained  for  several 
hours,  or  until  the  drug  is  eliminated  and  recovery  occurs. 


42 


EXPERIMENTAL   PHARMACOLOGY. 


PILOCARPINE. 

Experiments  Showing  the  Action  of  Pilocarpine.  PAGE. 

1.  On  the  frog , 42 

2.  On  the  mammal 42 

3.  On  the  ventricular  muscle   .    . .;.....     43 

4.  On  the  frog's  heart 43 

5.  On  the  circulatory  and  respiratory  systems  of  the  mammal  .     43 

i.  Pilocarpine  on  the  frog.  Give  a  frog  a  hypodermic  injection 
of  0.6  c.c.  of  10  percent  solution  of  pilocarpine  nitrate.  Keep  the 
frog  in  a  moist  battery  jar  until  normal  again.  The  toxic  dose  is  I  c.c.  of 
10  percent  solution  of  pilocarpine. 

"on       5#~~ 


FIG.  24. — Blood-pressure  and  respiratory  effects  of  an  intravenous  injection  of  i 
c.c.  of  i  percent  pilocarpine  in  the  dog.  Injection  between  the  points  "on"  and 
"off."  Time  in  seconds.  Pressure  in  mercury.  Reduced  to  four-fifths  the  original  size. 

2.  Pilocarpine  on  the  mammal.  Give  a  dog  a  hypodermic  injection 
of  0.3  c.c.  (5  minims)  i  percent  pilocarpine.  This  dose  produces  a 
marked  secretion  by  glandular  structures.  Examine  the  flow  of  saliva  by 
turning  back  the  upper  lip,  drying  it  and  noting  the  accumulation  of  drops 


PILOCARPINE.  43 

at  the  mouth  of  the  salivary  duct.     An  injection  of  i  c.c.  of  o.i  percent 
atropine  in  a  vein  antagonizes  pilocarpine  and  stops  the  secretion. 

Give  additional  drops  of  I  percent  pilocarpine  in  the  eye.     Drops 
of  i  percent  atropine  on  the  eye  will  overcome  the  action. 

3.  Pilocarpine  on  the  ventricular  muscle.     Mount  a  strip  of  the 
terrapin's   ventricle  in   physiological  saline.     When   the  contractions  are 
regular  transfer  to  a  o.i  percent  pilocarpine  solution  in  saline.     Allow  it 
to  act  only  i  minute  then  renew  the  physiological  saline  bath.     As  a  final 
test  give  the  strip  a  continuous  bath  of  o.i  percent  pilocarpine  and  note 
the  successive  effects. 

4.  Pilocarpine  on  the  frog's  heart.     Pith  a  frog,  expose  the  heart 
and  take 'tracings  of  the  ventricle.     Test  the  activity  of  the  vagus  with  a 
strong  interrupted  current.     Now  irrigate  the  surface  of  the  heart  with 
drops  of  i  percent  pilocarpine  for  two  minutes.    Stimulate  the  vagus  trunk 
one  minute  after  pilocarpine  and  at  successive  intervals  of  five  minutes.    If 
the  drug  is  strongly  active  the  heart  will  beat  slower.     In  the  early  stages 
the  stimulation  will  result  in  acceleration,  but  in  no  inhibition  as  in  the 
normal.     Applying  the  electrodes  directly  to  the  sinus  gives  no  inhibition 
showing  that  the  pilocarpine  has  acted  on  the  vagus  endings  and  not  on  the 
ganglionic  connections.     Drops  of  i  percent  atropine  sulphate  will  restore 
the  heart  beat  after  pilocarpine,  these  two  drugs  being  antagonists. 

5.  Pilocarpine  on  the  circulatory  and  respiratory  system  of  the 
mammal.     Anesthetize  a  dog  with  morphine  and  chloroform.     Introduce  a 
tracheal  canula  and  be  prepared  to  use  artificial  respiration  if  necessary. 
Take  blood-pressure  and  respiration  records  on  a  continuous  paper  kymo- 
graph.    Test  the  activity  of  the  vagus.     Give  an  intravenous  injection  of 
i  c.c.  of  i  percent  pilocarpine  nitrate.     Retest  the  activity  of  the  vagus 
after  the  pilocarpine.     Atropine  antagonizes  the  pilocarpine  effect.     Ex- 
amine the  pupils  from  time  to  time.     Also  note  the  increased  rate  of  salivary 
secretion. 


44  EXPERIMENTAL    PHARMACOLOGY. 


PHYSOSTIGMINE. 

Experiments  on  the  Action  of  Physostigmine.  PAGE. 

1.  On  the  frog 44 

2.  On  cardiac  muscle 44 

3.  On  the  frog's  heart  and  its  nervous  apparatus 44 

4.  On  the  heart  of  the  cat 44 

5.  On  the  respiratory  medullary  center  and  on  the  circulatory 

system  of  the  mammal 44 

6.  On  the  eye 47 

1.  Physostigmine  on  the  frog.     Give  a  frog  a  dorsal  lymph  sac 
injection  of  i  c.c.  (17  minims)   i  percent  physostigmine.     The  effects 
produced  are  diminished  irritability,  loss  of  muscular  tone,  paralysis  of  the 
respiratory  center,  loss  of  reflexes,  and  death,  or,  at  best,  a  very  slow  and 
prolonged  recovery. 

2.  Physostigmine   on   cardiac  muscle.     After   a   ventricular  strip 
from  the  terrapin  has  begun  beating  regularly  in  physiological  saline,  transfer 
it  to  o.i  percent  physostigmine  in  saline  for  two  to  three  minutes.     Physi- 
ological   saline  recovers   the  normal   contractions    after   several   minutes. 
Compare  the  results  with  those  from  pilocarpine  and  muscarine.     A  bath  of 
0.002  percent  atropine  antagonies  the  physostigmine  effects. 

3.  Physostigmine  on  the  frog's  heart  and  its  nervous  apparatus. 
Pith  a  frog  and  take  a  record  of  the  heart  beat.     Determine  the  minimal 
effective  stimulus  of  the  vagus  trunk  for  inhibition  of  the  heart  rate.     Now 
irrigate  the  surface  of  the  heart  with  drops  of  o.i  percent  physostigmine 
from  an  irrigating  bottle  for  two  minutes.    Redetermine  the  minimal  stimulus 
for  the  vagus  trunk  beginning  with  a  very  weak  induction  current.     If 
the  contractions  are  at  a  slow  rate  or  have  ceased,  irrigate  the  surface  of  the 
heart   with    i    percent   atropine.     Atropine   antagonizes   physostigmine. 
Compare  with  pilocarpine.     If  the  perfusion  method  is  used  then  o.oi 
percent  physostigmine  is  the  proper  solution  strength  for  the  frog's  heart. 

4.  Physostigmine  on  the  isolated  heart  of  the  cat.    Prepare  an 
isolated  cat's  heart  by  the  method  used  in  the  nicotine  experiment  5.     When 
it  gives  regular  contractions,  perfuse  it  with  o.oi  percent  physostigmine 
in  Locke-blood  solution. 

5.  Physostigmine  on  the  respiratory  medullary  center  and  on 
the  circulatory  system  of  the  mammal.     Anesthetize  a  dog,  insert  a  trach- 


PHYSOSTIGMINE. 


45 


EXPERIMENTAL    PHARMACOLOGY. 


eal  canula  and  be  prepared  for  artificial  respiration.  Take  a  continuous 
record  of  the  blood-pressure  and  of  the  respiratory  movements.  Insert  a 
canula  in  the  saphenous  vein  and  connect  it  with  a  buret  containing  the  drug. 
Give  an  intravenous  injection  of  o.i  percent  physostigmine  slowly  until  the 
first  effects  are  noticed  on  blood-pressure.  Note  the  amount  and  mark 
the  time  of  the  dose  on  the  record  by  a  signal  pen.  Usually  there  is  a  pro- 

1 


njuuuijuuuuijuuuuuuuuuuuu^ 


FIG.  26. 

FIG.  26. — Action  of  physostigmine  and  its  antagonism  by  atropine  in  the  mammal, 
showing  the  effects  of  an  injection  of  8  mgr.  of  physostigmine  just  before  the  part  of 
of  respiration  and  the  circulation  effects  upon  the  injection  of  2  mgr.  of  atropine  be- 
At  a,  b  and  c  are  shown  portions  of  the  trace  at  30-second  intervals.  Time  in  seconds. 

gressive  paralysis  of  the  respiratory  center  accompanied  by  a  great  slowing 
of  the  heart  and  a  fall  of  blood-pressure  by  one-half  or  more.  The  heart 
continues  to  beat  long  after  respiration  ceases.  Use  artificial  respiration 
until  the  blood-pressure  improves  and  the  anesthesia  becomes  light.  This 
will  not  restore  automatic  respiratory  movements  as  it  does  after  heavy 
anesthesia.  If  at  this  tinie  a  venous  injection  of  0.5  c.c.  of  i  percent 


PHYSOSTIGMINE. 


47 


atropine  be  given  from  a  hypodermic  syringe  the  respiratory  movements 
will  be  quickly  established  and  the  slow  heart  and  low  pressure  will  give 
way  to  the  rapid  heart  and  strong  pressure  following  primary  injection  of 
atropine,  experiment  6,  page  35.  The  vagus  inhibitory  apparatus  is  effective 
under  physostigmine,  but  not  after  atropine.  Repeat  the  experiment.  It 
takes  a  larger  dose  of  physostigmine  to  overcome  the  atropine  and  pro- 


a— L 


.....    . 


. 


FIG.  26  (Continued). 

Parts  i,  iX  and  iT  give  the  respiration  trace,  blood-pressure  and  time,  respectively, 
the  trace  shown.  The  parts  of  the  trace  numbered  2,  2X  and.2T  show  the  recovery 
tween  the  arrows.  A  few  second's  gap  intervenes  between  the  two  parts  of  the  figure. 


duce  the  characteristic  effects.     Examine  the  pupil  before  and  after  the 
physostigmine. 

6.  Physostigmine  on  the  eye.  Give  2  drops  of  i  percent  phy- 
sostigmine in  one  eye  of  a  dog  or  a  rabbit,  at  intervals  of  five  minutes.  It 
is  better  to  use  one  of  the  experimeter's  own  eyes.  Strong  contraction  of 
the  pupil  follows.  A  decrease  in  intraocular  pressure  has  also  been  proven, 


48  EXPERIMENTAL    PHARMACOLOGY. 

'i 

and  to  produce  this  effect  is  the  chief  therapeutic  use  of  the  drug.  A  strik- 
ing comparison  is  obtained  by  dropping  i  percent  atropine  in  the  unused 
eye  of  the  dog  after  the  physostigmine  effect  has  come  on  in  the  other  eye. 
Physostigmine  will  overcome  the  atropine  dilation  of  the  pupil.  The  experi- 
menter may  show  the  antagonism  between  homatropine  and  physostigmine 
on  his  own  eyes,  but  it  is  recommended  that  one  eye  always  be  reserved. 


ACONITE. 

Experiments  on  the  Action  of  Aconite.  PAGE. 

1.  On  the  frog j. ;~-\.    ....     48 

2.  On  the  circulatory  system  of  a  mammal.    .    .  v\V  ....     48 

3.  On  the  frog's  heart :'  V   ....     48 

4.  On  the  mammalian  heart 48 

1.  Aconite  on  the  frog.    The  dose  is  0.5  c.c.  of  o.i  percent  aconite. 

Compare  with  digitalis. 

2.  Aconite  on  the  circulatory  system  of  the  mammal.     Take  a 
continuous  tracing  of  the  blood-pressure  of  a  dog.     Give  I  c.c.  of  o.i 
percent  aconitine  crystals.     Note  particularly  the  progressive  effects  on 
the  nervous  and  muscular  elements  of  the  circulatory  apparatus. 

3.  Aconite  on  the  frog's  heart.     Destroy  the  cerebrum  and  optic 
lobes  only  of  a  frog,  expose  the  ventricle  and  take  a  tracing.     Give  an  in- 
jection of  0.5  c.c.  of  o.i  percent  aconitine  in  the  lymph  sac.     One  may 
expect   a   progressive   stimulation   of   the  accelerator  and  vagus  nervous 
apparatus  followed  by  paralysis  of  nerves  and  muscle. 

4.  Aconite  on  the  mammalian  heart.    Prepare  an  isolated  heart 
as  described  for  the  nicotine  experiment  5.     When  the  heart  is  contracting 
regularly  with  the  Locke-blood  perfusion  then  perfuse  for  10  seconds  with  a 
0.0002  percent  aconite  solution.     A  prolonged  perfusion  or  perfusion 
with  a  stronger  concentration  of  aconite  will  quickly  set  up  incoordinate 
contractions  and  fibrillation. 


ACONITE. 


49 


5O  EXPERIMENTAL    PHARMACOLOGY. 

VERATRINE. 

Experiments  on  the  Action  of  Vera trine.  PAGE. 

1.  On  the  frog 50 

2.  On  the  mammal 50 

3.  Veratrine  on  heart  strip.    . 50 

4.  On  the  frog's  heart. 50 

5.  On  the  isolated  mammalian  heart 50 

6.  On  the  form  of  the  simple  muscle  contraction 50 

7.  On  the  circulatory  and  respiratory  systems  of  the  mammal. .  50 

1.  Veratrine  on  the  frog.     The  dose  for  a  frog  is  about  0.5  c.c. 
of  a  i  percent  solution  of  the  fluid  extract  veratrum  viride  or  0.3  c.c.  of 
o .  o  i  percent  vera trine .     Compare  with  the  effects  of  aconite  and  of  barium. 
See  experiment  4. 

2.  Veratrine  on  the  mammal.     Give  a  cat  or  rabbit  i  c.c.  of  o.i 
percent  veratrine  hypodermically,  or  i  c.c.  of  i  percent  for  a  dog.     Keep 
under  observation  for  a  considerable  time. 

3.  Veratrine  on  the  heart  strip.     Subject  the  contracting  strip  of 
ventricle  to  0.005  to  0.05  percent  vera  tine  in  saline. 

4.  Veratrine  on  the  frog's  heart.     Pith  a  frog,  expose  the  heart  and 
take  a  tracing  when  perfused  with  0.005  percent  veratrine  in  Ringer's 
solution  (o.oi  percent  destroys  coordination). 

5.  Aconite  on  the  isolated  mammalian  heart.    Prepare  the  appara- 
tus for  the  isolated  heart  experiment,  isolate  a  cat's  heart  and  perfuse  with 
0.0002  percent  veratrine  in  Locke-blood  solution.     See  Fig.  25. 

6.  Veratrine  on  the  simple  muscle  contraction  of  the  frog.     Ligate 
one  leg  of  a  frog  and  give  a  hypodermic  of  0.5  c.c.  of  o.i  percent  vera- 
trine.    After  15  minutes  prepare  the  veratrinized  muscle  and  take  simple 
muscle  contractions  to  show  the  form  of  the  contraction  wave,  using  a 
tuning  fork  to  record  the  drum  speed.     Compare  this  curve  with  that  of 
the  undrugged  muscle. 

The  frog  of  experiment  i  may  be  used  to  show  the  veratrine  effect  on 
muscle  work.  Stimulate  once  in  three  seconds  in  this  experiment,  since  the 
relaxation  may  not  be  complete  in  an  interval  of  two  seconds. 

7.  Veratrine  on  the  circulation  and  respiration  of  a  mammal. 
Take  a  record  of  the  blood-pressure  from  the  carotid  of  an  anesthetized  dog. 
Tracheotomize  and  take  respiratory  tracings.     Give  i  c.c.  of  I  percent 
veratrine  in  the  abdominal  cavity.     When  marked  cardiac  slowing  appears 
cut  the  vagi  and  note  the  effects  on  the  heart. 


VERATRINE. 


o-    O 


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52  EXPERIMENTAL    PHARMACOLOGY. 


DIGITALIS. 

7 

Experiments  Showing  the  Action  of  Digitalis.  PAGE. 

1.  On  the  frog.    ...'.• .    .    .    .  52 

2.  On  the  ventricular  muscle ...,..;....  52 

3.  On  the  frog's  heart ....'. 52 

4..  On  the  atropinized  frog's  heart ;. /.'.«..-.    ...  52 

5.  On  the  mammalian  heart -":    .'....  52 

6.  On  the  circulatory  and  respiratory  systems  of  the  mammal.  53 

7.  Digitalis  as  a  diuretic t.  •. ,,.-„    ....  53 

1.  Digitalis  on  the  frog.     Give  a  dose  of  0.5  c.c.  of  0.2  percent 
of  soluble  digitalis.     The  digitalis  effects  develop  slowly.     Note  the  heart 
rate,  and  particularly  the  circulation  in  the  web.     Keep  in  a  moist  battery 
jar  for  at  least  two  hours.     Examine  the  heart  if  death  occurs. 

2.  Digitalis  on  the  ventricular  muscle.     Treat  a  strip  of  terrapin's 
ventricle  contracting  in  saline  to  a  bath  of  0.002  percent  digitalis  in  saline. 
Follow  with  pure  saline.     Repeat  with  a  0.005  percent  digitalis.     Still 
stronger  solutions  may  be  used,  but  a  marked  tone  will  result  as  shown  in 
Figure  26. 

Digitalis  solutions  may  be  used  made  up  in  the  weaker  Ringer,  but 
as  the  rate  is  slower  and  the  amplitude  much  greater  than  that  in  sodium 
chloride  solutions  the  picture  will  be  quite  different,  though  the  same  in 
kind.  Delirium  cordis  of  the  strip  is  produced  by  the  stronger  solutions  act- 
ing for  several  minutes. 

3.  Digitalis  on  the  frog's  heart.     Pith  a  frog  and  take  a  record  of 
the  contractions  of  the  ventricle  when  irrigated  with  physiological  saline. 
Irrigate  slowly  with  drops  of  0.2  percent  digitalis  for  two  minutes,  then 
wash  off  with  saline. 

A  more  effective  method  is  to  perfuse  the  heart  from  a  canula  in  the 
vena  cava.  Use  a  niuch  weaker  solution  for  perfusion,  i.e.,  0.0005  to 
o.ooi  percent  digitalis  in  Ringer.  These  effects  should  be  compared  with 
the  effects  on  cardiac  muscle  above. 

4.  Digitalis  on  the  atropinized  frog's  heart.    Atropinize  the  frog's 
heart  to  eliminate  the  cardiac  nervous  control,  then  repeat  experiment  3 
above. 

5.  Digitalis   on   the  mammal  heart.     Use  the  method  described 
on  page  70.     Isolate  and  perfuse  the  cat's  heart  with  the  normal  solution 


DIGITALIS. 


53 


of  Locke-blood,  then  with  o.oooi  percent  digitalis  (soluble  digitalis) 
in  Locke-blood.  Increase  the  strength  to  0.0005  percent.  The  stronger 
solution  will  usually  produce  a  great  increase  in  amplitude  followed  by 
fibrillation.  The  weaker  solution,  see  figure  30,  produces  a  typical  mild 
therapeutic  effect  on  the  heart.  Compare  with  the  results  of  experiment  6. 
6.  Digitalis  on  the  circulatory  and  respiratory  systems  of  the 
mammal.  Anesthetize  a  dog  and  take  continuous  kymographic  records 
of  the  blood-pressure  and  of  the  respiration.  Slowly  inject  into  the  saphe- 
nous  vein  2  c.c.  doses  of  0.5  percent  digitalis  at  five-minute  intervals 


FIG.  29. — Experiment  showing  the  action  of  digitalis  on  the  rhythm  and  tone  of  a 
strip  of  terrapin's  ventricle.  The  strip  was  contracting  in  physiological  saline.  Between 
the  words  "on"  and  "off"  it  was  subjected  to  0.06  percent  of  digitalis  in  saline. 

until  the  three  stages  of  digitalis  effect  on  the  heart  and  blood-pressure  are 
obtained.  The  anesthetic  must  be  perfectly  constant.  One  may  give  the 
maximal  dose  of  4  c.c.  of  i  percent  digitalis  at  once.  In  this  instance  the 
three  stages  are  passed  through  rapidly  and  the  animal  will  usually  die  in 
10  to  20  minutes.  Read  Cushny's  Pharmacology,  pp.  430-435. 

7.  Digitalis  as  a  diuretic.  Morphinize  and  chloroform  a  dog. 
Take  the  blood-pressure.  Isolate  the  ureters  near  the  bladder  and  insert 
canulas,  using  care  not  to  occlude  the  ureters  by  twisting  or  otherwise.  Con- 
nect the  ureters  by  means  of  a  T-tube  with  a  horizontal  2  c.c.  pipet  graduated 


54 


EXPERIMENTAL    PHARMACOLOGY. 


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ERGOT.  55 

to  1/50  c.c.  Close  the  abdomen  with  sutures.  Insert  a  venous  canula  and 
connect  with  a  transfusion  buret.  Establish  the  normal  secretion  per  10 
minutes,  cutting  off  the  column  of  secreted  urine  by  injecting  a  bubble  of  air 
into  the  mouth  of  the  buret  by  inserting  a. hypodermic  needle  through  the 
rubber  connecting  tube.  Now  inject  5  c.c.  of  o.i  percent  digitalis  or 
strophanthin  into  a  vein  and  take  the  secretion  in  successive  10  minute 
periods  until  the  flow  is  constant.  Repeat  the  dose  once  or  twice  at  long  in- 
tervals. Mark  the  secretion  intervals  on  the  blood-pressure  record. 

Compare  these  results  with  those  observed  on  other  diuretic  drugs- 
caffeine,  urea,  inorganic  salts,  etc. 


ERGOT. 

Experiments  on  the  Action  of  Ergot.  PAGE. 

1.  On  the  frog 55 

2.  On  the  heart  muscle 55 

3.  On  the  arterioles  of  the  frog. 55 

4.  On  the  blood-pressure  and  heart  rate  of  a  mammal.     ...     55 

1.  Ergot  on  the  frog.     Give  0.5  c.c.  of  the  fluid  extract. 

2.  Ergot  on  the  heart  muscle.     Change  a  contracting  heart  strip 
from  saline  to  a  i  o  percent  solution  of  Squibbs'  fluid  extract  of  ergot  in  saline 
solution.     Allow  it  to  act  for  five  minutes.     Take  a  continuous  record. 

3.  Ergot  on  the  arterioles  of  the  frog's  web.    Wrap  a  frog  in 
a  wet  cloth  and  fasten  to  a  frog-board  for  examining  the   web.     Give  a 
lymph  sac  injection  of  0.5  c.c.  fluid  extract  of  ergot.     Select  a  good  field 
of    small    arterioles   and  measure  their  diameter  at  once.     The  relative 
change  in  diameter  of  small  vessels  can  be  determined  by  selecting  a  field 
in  which  pigment  spots  mark  the  borders  of  the  vessels.     Sketch  such  a 
vessel  and  spots  for  the  normal.     Re-sketch  after  the  drug.     Re-measure  at 
intervals  of  five  minutes  as  the  ergot  is  absorbed. 

4.  Ergot  on  the  blood-pressure  of  a  mammal.    Give  an  intravenous 
dose  of  0.5  c.c.  fluid  extract  of  ergot  to  a  mammal  while  taking  a  record 
of  the  blood-pressure. 


56  EXPERIMENTAL    PHARMACOLOGY. 


SUPRARENAL  GLAND. 

The  commercial  preparation  of  the  active  principle  of  suprarenal  gland, 
adrenalin  hydrochloride,  presents  the  same  physiological  action  as  the  gland 
extract  and  has  the  special  advantage  of  preparation  in  definite  and  known 
strengths.  It  has  come  into  general  use  for  therapeutic  purposes  and  is, 
therefore,  used  in  these  experiments. 

Experiments  Showing  the  Action  of  Adrenalin  Hydrochloride.  PAGE. 

1.  On  the  frog .  '.    .  56 

2.  On  the  ventricular  strip .......  56 

3.  On  the  frog's  heart 56 

4.  On  the  isolated  mammalian  heart :\"    .   ....  .    .  56 

5.  On  the  simple  muscle  contraction 'V   .  -.    .    .  58 

6.  On  muscle  work •.    .  v  .    .    .    .  58 

7.  On  the  local  mucous  surfaces .    . 58 

8.  On  the  size  of  the  blood-vessels  in  the  frog's  web  ....  58 

9.  On  general  blood-pressure  and  peripheral  vaso-constriction  58 

1.  Adrenalin  on  the  frog.     Give  0.5  c.c.  o.i  percent  in  the  dorsal 
lymph  sac. 

2.  Adrenalin   on   the   ventricular  muscle.     Transfer  a  terrapin's 
ventricular  strip  contracting  in  physiological  saline  to  o.oi  percent  adren- 
alin in  saline.     Change  after  two  to  five  minutes.     The  drum  speed  should 
be  i  cm.  per  minute.     Suprarenal  extract  has  also  been  shown  to  increase 
the  amplitude  and  the  rate  of  the  ganglion  free  ventricular  muscle  of  the 
dog.'     Cleghorn,  Amer.  Jour.  Physiol.,  Volume  III,  p.  273. 

3.  Adrenalin  on  the  frog's  heart.     Use  the  perfusion  method,  page 
68,  with  the  heart  in  place  and  the  inflow  canula  in  the  ascending  vena  cava. 
Follow  physiological    Ringer   perfusion   with    o.ooi    percent   adrenalin 
hydrochloride  in  Ringer.     The  drum  speed  should  be  2  mm.  per  second. 
Direct  application  to  the  surface  of  the  heart  requires  a  strength  of  at  least 
0.05  percent  adrenalin  hydrochloride. 

4.  Adrenalin  on  the  isolated  heart.     Perfuse  a  cat's  heart  in  the 
usual  way  with  Locke-blood  solution  for  a  normal,  then  change  to  a  o.oooi 
percent  adrenalin  hydrochloride  in  Locke-blood.     If  the  heart  be  beating 
feebly  it  often  happens  that  the  contractions  will  increase  in  amplitude  by 
200  percent  and  more. 


SUPRARENAL    GLAND. 


57 


58  EXPERIMENTAL    PHARMACOLOGY. 

5.  Adrenalin  on  the  simple  muscle  contraction.     Ligate  one  leg 
of  a  frog  and  give  0.5  c.c.  of  0.05  percent  adrenalin.     Allow  ten  minutes 
for  absorption.     Compare  the  simple  muscle  contractions  of  the  two  gastroc- 
nemii  as  repards  a,  amplitude,  and  b,  the  time  of  the  simple  contraction. 
The  muscular  power  of  patients  with  Addison's  disease  has  also  been  shown 
to  be  greatly  improved  by  giving  the  extract  of  suprarenal  gland. 

6.  Adrenalin  on  muscle  work  in   the  frog.    Prepare  a  frog  as  in 
experiment  5  above  and  test  the  work  performed  by  the  two  muscles.     For 
details  of  procedure  see  alcohol  experiment  5. 

7.  Adrenalin  on  mucous  surfaces.     Paint  one-half  the  tongue  with 
o.i  percent  adrenalin  hydrochloride.     At  intervals  of  five  minutes  drop 
o.oi  percent  in  saline  (sterilize  by  boiling)  in  one  eye.     Compare  the  two 
halves  of  the  tongue  and  the  two  eyes  as  to  vascular  condition.     Examine 
the  size  of  the  pupils.     Test  for  possible  differences  as  to  the  sensitiveness 
of  the  conjunctiva.     Try  the  effect  in  the  eye  of  a  cat  or  dog. 

8.  Adrenalin  on  the  size  of  the  blood-vessels  of  the  frog's  web. 
Use  a  dose  of  0.5  c.c.  of  o.i  percent  as  a  hypodermic.     See  ergot  experi- 
ment 3,  page  55;  nitroglycerine  experiment  3,  page  59.     Or  apply  drops  of 
o.i  percent  directly  to  the  web  under  the  microscope. 

9.  Adrenalin  on  general  blood-pressure  and  on  vaso-constriction 
in  a  mammal.     Prepare  a  dog  for  blood-pressure.     Adjust  an  onkometer 
to  the  left  kidney  and  record  the  change  in  volume  with  a  Brodie's  bellows 
or  Roy's  piston  recorder.     Give  2  to  4  c.c.  of  o.oi  percent  adrenalin 
hydrochloride  slowly  in  a  vein.     Give  2  c.c.  of  o.i  percent  atropine  to 
eliminate  the  vagus  action  on  the  heart  and  repeat  the  adrenalin.     Compare 
with    digitalis,  page   53;    ergot,   page   55;  vera trine,   page   50.     Drugs  of 
antagonistic  action  are  nitrites  and  potash  salts. 


NITROGLYCERINE    AND    THE*  NITRITES.  59 

NITROGLYCERINE  AND  THE  NITRITES. 

Experiments    on    the    Action    of    Nitroglycerine    and    the 

Nitrites.  PAGE. 

1.  On  the  frog 59 

2.  On  the  heart  muscle 59 

3.  On  the  arterioles  of  the  frog 59 

4.  On  the  circulation  volume 59 

5.'  Amyl  nitrite  on  the  pulse 59 

6.  Nitrites  on  mammalian  blood-pressure 60 

Nitroglycerine  and  the  nitrites  affect  primarily  the  peripheral  circu- 
lation, causing  vaso-dilation  with  fall  of  blood-pressure.     The  specific  action 
is  on  the  muscular  tissue. 

1.  Nitroglycerine  on  the  frog.     Give  a  frog  a  dose  of  0.5  c.c.  of 
o.i  percent  nitroglycerine  in  the  dorsal  lymph  sac. 

2.  Sodium  nitrite  on  the  heart  muscle.     Test  the  action  of  0.02 
percent  sodium  nitrite  on  the  contracting  ventricular  strip. 

3.  Nitroglycerine  on  the  arterioles  of  a  frog.     Bind  a  frog  for  the 
microscopic  examination  of  the  web  circulation.     Then  give  I  c.c.  of  o.i 
percent   nitroglycerine   in   the   lymph   sac.     Immediately   measure   the 
smaller  arterioles  in  a  favorable  field  and  re-examine  every  two  minutes  as 
absorption  progresses.     Try  direct  application  of  drops  of  o.i  percent  to  the 
web. 

4.  Sodium  nitrite  on  the  circulation  volume.    Pith  a  frog  or  small 
terrapin.     Insert  a  canula  in  the  aorta   or  one  of  its  branches,  snip  the 
veins  with  the  scissors  to  allow  free  perfusion,  set  the  frog-board  at  an  angle 
to  facilitate  drainage  of  liquid.     Perfuse  the  blood-vessels  with  a  weaker 
Ringer's  solution  for  a  normal.    Follow  with  o.oi  percent  sodium  nitrite 
in  the  weaker  Ringer,  keeping  a  uniform  pressure  of  the  perfusion  liquids 
of  from  6  to  10  cm.     Measure  the  perfusion  rate  in  drops,  per  minute,  or 
collect  the  outflow  in  a  25  c.c.  graduate. 

Test  the  amount  of  outflow  when  irrigated  with  0.0005  percent  of 
soluble  digitalis,  then  follow  with  o.oooi  percent  sodium  nitrite,  both  in  the 
weaker  Ringer. 

5.  Amyl  nitrite  on  the  pulse.     Take  normal  pulse  records  with  one  of 
the  standard  sphygmographs.     Break  an  amyl  nitrite  pearl  on  a  handkerchief 
and  breathe  deeply  the  fumes.     Pulse  tracings  taken  5  and  10  minutes  later 
will  show  the  usual  signs  of  dilated  blood-vessels  with  accompanying  low 
pressure.     Slight  headaches  sometimes  follow  the  use  of  amyl  nitrite. 


6o 


EXPERIMENTAL  PHARMACOLOGY. 


6.  Nitrites  on  mammalian  blood-pressure.  Anesthetize  a  10  kilo 
dog  and  take  the  blood-pressure.  Give  intravenous  doses  of  nitrites  in  the 
following  order,  repeating  with  larger  doses  if  necessary  and  always  allowing 
full  time  for  recovery:  i  c.c.  of  o.i  percent  nitroglycerine,  3  c.c.  of  o.i 
percent;  2  c.c.  of  o.i  percent  amyl  nitrite;  6  c.c.  of  o.i  percent 


FIG.  32. — Action  of  amyl  nitrite  on  the  human  pulse.  One  amyl  nitrite  pearl  was 
crushed  on  a  handkerchief  and  the  fumes  inhaled  deeply.  Trace  i  is  the  normal  pulse. 
Trace  2  immediately  after  amyl  nitrite  fumes.  Traces  3  and  4  are  stages  of  recovery. 
Time  in  seconds. 

sodium  nitrite.  The  blood-pressure  remains  low  for  a  long  time  after 
sodium  nitrite.  A  dose  of  2  c.c.  to  5  c.c.  of  0.2  percent  digitalis  or  2  c.c. 
of  o.oi  percent  adrenalin  hydrochloride  will  antagonize  this  effect. 

Give  2  c.c.  of  o.i  percent  atropine  to  eliminate  the  action  of  the  car- 
diac nervous  apparatus  and  repeat  the  above  doses  of  nitroglycerine  and 
sodium  nitrite. 


CARBOLIC    ACID.  6l 


CARBOLIC  ACID. 

Experiments  on  the  Action  of  Carbolic  Acid.  PAGE. 

1.  On  the  frog 61 

2.  On  the  growth  of  yeast  and  bacteria 61 

3.  On  the  circulatory  and  respiratory  systems  of  a  mammal     61 

1.  Carbolic  acid  on  the  frog.     Give  a  dose  of  i  c.c.  of  i  percent. 

2.  Carbolic  acid  on  the  growth  of  yeast  and  of  bacteria.    Prepare 
six  fermentation  tubes  of  active  yeast  culture  and  as  many  test-tubes  of 
inoculated  bouillon.     Keep  one  tube  of  each  for  a  normal  and  to  the  others 
add  enough  10  percent  carbolic  acid  to  make  a  series  of  o.i,  0.5,  i,  2,  and 
4  percent  solutions.     Keep  at  laboratory  temperature  and  observe  through  a 
period  of  several  days. 

3.  Carbolic  acid  on  the  circulatory  and  respiratory  systems  of  the 
mammal.     While  taking  records  of  blood-pressure  and  respiration  by  the 
usual  method  give  an  intravenous  injection  of  10  c.c.  of  0.5  percent  carbolic 
acid.     When  the  collapse  stage  is  far  advanced  inject  i  percent  sodium 
sulphate  slowly.     Judge  the  amount  required  by  the  action  in  overcoming 
the  carbolic  acid  depression  of  the  respiratory  apparatus. 

POTASSIUM  SALTS. 

Experiments  Showing  the  Action  of  Potassium  Salts.  PAGE. 

1.  On  the  heart  muscle 61 

2.  On  the  reaction  time  in  the  reflex  frog 61 

3.  On  muscle  irritability  and  muscle  work  in  the  frog.    .    .    .     61 

1.  Potassium  chloride  on  the  heart  muscle.     A  ventricular  strip 
contracting  in  physiological  saline  solution  is  transferred  to  0.06  percent 
potassium  chloride  in  saline  for  two  to  five  minutes.     Contractions  return 
in  saline  even  after  stronger  doses  of  potash. 

2.  Potassium  bromide  on  the  reaction  time  in  the  reflex  frog. 
Compare  the  reaction  time  of  a  reflex  frog  before  and  20  to  40  minutes  after 
0.3  c.c.  of  5  percent  potassium  bromide  in  the  dorsal  lymph  sac. 

3.  Potassium  chloride  on  muscle  irritability  and  muscle  work 
in  the  frog.     Compare  the  two  gastrocnemii  as  to  irritability  and  as  to 
amount  of  muscular  work  done.     Dose  0.3  c.c.  of  5  percent  solution  given 
hypodermic  after  one  leg  is  ligatured. 


62 


EXPERIMENTAL    PHARMACOLOGY. 


CALCIUM  SALTS. 

Experiments  Illustrating  the  Action  of  Calcium  Salts.  PAGE. 

1.  On  heart  muscle    .    .    .    .......    .    .    .    .    .'.    .    .    .  62 

2.  On  the  frog's  heart       .    ...    .    .    .    .    , .  62 

3.  On  the  blood-pressure  and  the  respiration  in  the  mammal.  63 

i.  Calcium  chloride  on  heart  muscle.  Transfer  a  ventricular  strip 
from,  physiological  saline  to  0.03  percent  calcium  chloride  in  saline  for 
three  to  five  minutes.  Record  on  a  drum  speed  of  2  cm.  per  minute. 
Repeat,  using  0.06  percent.  The  rate  is  increased  and  the  amplitude  often 


FIG.  33. — Terrapin's  heart  muscle  as  influenced  by  a  solution  of  0.06  percent  cal- 
cium chloride  in  physiological  saline.  A  weaker  solution  produces  much  less  tone. 
Time  in  seconds. 

doubled.  The  stronger  solution  produces  great  increase  in  tone  which 
sometimes  passes  into  delirium  cordis.  Potash  salts  antagonize.  Read 
Ringer,  Jour.  Physiology,  1883. 

2.  Calcium  chloride  on  the  frog's  heart.  Perfuse  the  frog's  heart 
through  the  vena  cava  with  0.7  percent  sodium  chloride  and  follow  with 
0.03  percent  calcium  chloride  in  0.7  percent  sodium  chloride.  Recover 


BARIUM    SALTS.  63 

the  sodium  chloride  type  of  contractions,  then  perfuse  with  o.oi  percent 
barium  in  sodium  chloride. 

3.  Calcium  chloride  on  the  blood-pressure  and  the  respiration 
in  the  mammal.  Give  an  intravenous  dose  of  10  c.c.  of  i  percent  for  a 
dog.  Cut  the  vagi  and  repeat  the  dose.  Alternate  the  dose  with  potassium, 
2  to  4  c.c.  of  i  percent. 


BARIUM  SALTS. 

Experiments  on  the  Action  of  Barium  Salts.  PAGE. 

1.  On  the  frog 63 

2.  On  the  heart  muscle 63 

3.  On  the  circulation  and  on  the  respiration  movements  in 

mammals     .    . 63 

1.  Barium  on  the  frog.     Dose,  i  c.c.  of  i  percent  barium  chloride. 

2.  Barium  chloride  on  the  heart  muscle.     Transfer  a  contracting 
ventricular  strip  from  0.7  percent  sodium  chloride  to  o.oi  percent  barium 
chloride  in  saline.     Short  immersions  increase  the  rate,  but  long  baths  show 
that  this  salt  does  not  sustain  contractions  as  do  calcium  salts.     A  o.i  percent 
solution  in  saline  delays  contractions  with  prevention  of  relaxation.     Con- 
tractions still  take  place  in   i   percent  barium  chloride.     Compare  with 
digitalis. 

3.  Barium  chloride  on  the  circulation  and  on  respiratory  move- 
ments in  mammals.     The  effect  on  the  heart  and  blood-pressure  and  on 
respiration  in  a  mammal  is  demonstrated  by  an  intravenous  dose  of  5  c.c. 
of  0.2  percent  given  slowly.     This  dose  should  be  repeated  several  times 
both  before  and  after  section  of  the  vagi.     Barium  salts  act  as  strong  poisons 
to  the  nerve  centers,  especially  those  in  the  medulla. 


OPERATIONS,  APPARATUS  AND  SPECIAL 

METHODS. 

PHYSIOLOGICAL  SOLUTIONS. 

The  lymph  and  blood  plasma  in  which  the  tissues  develop  are  the  true 
physiological  solutions. 

Artificial  solutions  imitate  lymph  in  its  isotonicity — its  physical  character, 
and  in  its  composition — its  chemical  character.  Sodium  chloride  in  0.6  per- 
cent solution,  used  first  by  Nasse  in  1869  on  frog's  muscle,  and  by  Bow- 
ditch  in  1871  on  the  frog's  heart,  was  supposed  to  prevent  injurious  changes 
in  the  tissue  by  virtue  of  its  isotonicity.  Ringer  in  1883  and  Locke  in  1885 
introduced  the  solutions  which  bear  their  names.  They  showed  that  the 
chemical  factors  play  a  fundamental  part  in  the  effects  of  these  solutions 
on  the  tissues.  At  the  present  time  we  recognize  that  exact  isotonicity  is  not 
nearly  so  fundamental  as  at  first  supposed,  and  that  these  solutions  are 
chemically  active  in  relation  to  the  living  protoplasm. 

At  the  present  time  much  attention  is  being  given  to  the  effects  of 
asphyxiation  on  the  physiological  activity  of  living  tissues  that  are  isolated 
from  the  normal  circulation.  The  artificial  solutions  can  be  made  more 
efficient  by  shaking  with  air  before  using,  or  by  shaking  with  pure  oxygen. 
Isolated  mammalian  hearts  give  much  more  constant  characters  in  their 
response  to  artificial  solutions  which  contain  defibrinated  blood,  prefer- 
ably from  the  animal  supplying  the  heart.  Even  with  five  to  ten  percent  of 
blood  such  solutions  are  strikingly  more  efficient,  supposedly  because  they 
are  much  better  oxygen  carriers.  For  cats'  hearts  Locke-blood  solutions 
aerated  by  a  stream  of  oxygen  are  very  efficient  indeed. 

1.  Physiological  salt  solution  or  normal  saline.     Sodium  chloride 
in  distilled  water  0.7  percent.     More  exact  isotonicity  is  secured  by  0.6 
percent  for  frogs,  0.7  percent  for  terrapin  and  0.9  percent  for  mammals. 

2.  Ringer's  solution.     The  Ringer's  solution  that  imitates  the  blood 
serum  in  its  effects  on  heart  tissue  is  made  up  in  this  laboratory  in  the 
following  proportions: 

Sodium  chloride,  0.7  percent. 
Potassium  chloride,  0.03  percent. 

64 


ANESTHESIA.  65 

Calcium  chloride  (cryst.  computed  water  free),  0.026  percent. 
For  heart  work  where  a  more  rapid  rate  is  desired  the  amount  of  potas- 
sium must  be  reduced  to  that  in  Ringer's  original  formula. 
Sodium  chloride,  0.7  percent. 
Potassium  chloride,  o.oi  percent. 
Calcium  chloride  (cryst.  computed  water  free),  0.026  percent. 

3.  Locke's  solution.     Locke's  solution  is  a  mixture  of  the  salts  in 
Ringer's  solution  with  dextrose  added  to  make  o.i  percent. 

Sodium  chloride,  0.7  percent. 
Potassium  chloride,  0.03  (or  o.oi)  percent. 
Calcium  chloride  (cryst),  0.026  percent. 
Dextrose,  o.i  percent. 

4.  Locke-blood  solution.     Add  5  to  10  percent  of  defibrinated  whole 
blood  to  the  above  Locke's  solution. 


ANESTHESIA. 

The  mammals  usually  available  for  laboratory  experimental  purposes 
are  dogs,  cats,  rabbits  and  guinea-pigs,  each  of  which  can  best  be  anesthe- 
tized by  a  special  treatment  of  its  own. 

Dogs.  Give  a  zo-kilo  dog  i  c.c.  (17  minims)  of  2  percent  mor- 
phine under  the  skin  of  the  shoulder,  holding  its  head  firmly  between  the 
operator's  legs  while  the  hypodermic  injection  is  being  given.  Allow  15 
minutes  or  more  for  the  morphine  to  take  effect.  The  morphine  should  be 
followed  by  chloroform  or  chloroform  and  ether  in  equal  parts.  Give  it 
by  means  of  a  small  nose  hood  made  by  sewing  a  cheese-cloth,  that  has  been 
folded  in  the  form  of  a  blunt  cone,  to  a  wire  ring,  or  use  a  Senn's  inhaler 
mask.  When  the  voluntary  movements  have  about  ceased,  tie  the  dog  to  a 
holder  and  take  it  to  the  experimental  table.  The  tests  of  good  anesthesia 
are:  i,  loss  of  voluntary  movements;  2,  no  cutaneous  reflexes;  3,  slight 
corneal  reflexes  or  none  in  deep  anesthesia ;  4,  even  and  fairly  deep  respira- 
tion; 5,  medium  blood-pressure  and  pulse.  This  condition  of  anesthesia 
is  maintained  by  giving  chloroform  from  a  dropping-bottle  at  abso- 
lutely regular  intervals  of  30  seconds  by  the  watch.  The  number  of 
drops  necessary  for  each  animal  will  quickly  be  found  by  trial.  In  the 
experience  of  this  laboratory  it  is  from  3  to  6  drops  per  30  seconds.  The 
success  of  most  pharmacological  experiments  on  dogs  depends  upon  main- 
taining an  absolutely  even  anesthesia. 

Cats.  A  mixture  of  equal  parts  of  chloroform  and  ether  is  the  most 
5 


66  EXPERIMENTAL    PHARMACOLOGY. 

practical  anesthetics  for  cats.  These  animals  are  anesthetized  most  conven- 
iently by  putting  them  in  a  box  of  about  two  cubic  feet  in  dimension  and 
provided  with  a  close  cover.  A  very  convenient  box  is  the  tin  display 
cracker  box  with  glass  window  obtained  of  the  grocer.  Drop  in  the  box  with 
the  cat  a  small  strip  of  cheese-cloth  saturated  with  chloroform-ether  mix- 
ture, 10  c.c.  in  broken  doses  will  anesthetize  a  cat  in  10  minutes.  As 
soon  as  the  animal  falls  down  under  the  influence  of  the  anesthetic  it 
should  be  taken  from  the  box,  fixed  in  the  holder,  and  the  anesthetic  given 
from  a  cloth  in  the  manner  and  with  care  prescribed  above  for  the  dog. 
Cats  do  not  survive  pure  chloroform  in  the  hands  of  the  ordinary  student 
anesthetist. 

Rabbits.  Give  rabbits  2  grams  of  urethane  by  the  mouth.  Follow 
with  light  and  careful  use  of  ether.  Or  pure  ether  may  be  given  without  the 
urethane.  Give  the  ether  in  the  manner  and  with  the  regularity  recom- 
mended above  for  giving  chloroform  to  dogs.  Do  not  use  chloroform  or 
even  chloroform  mixtures  with  rabbits. 

Guinea-pigs.  These  little  animals  when  they  must  be  used  for 
pharmacological  purposes  are  anesthetized  best  with  pure  ether  or  ether 
followed  with  a  little  morphine. 

THE  PREPARATION  OF  THE  VENTRICULAR  MUSCLE. 

Destroy  the  brain  of  a  terrapin,  remove  the  plastron  and  open  the  peri- 
cardium. Grasp  the  left  angle  of  the  base  of  the  exposed  ventricle  with  a 
forceps  and  cut  with  a  scissors  from  this  point  around  the  apex  to  the  oppo- 
site side,  thus  removing  a  piece  about  one  inch  long  and  the  size  of  the  half 
of  a  small  lead  pencil.  Split  this  strip  into  two  or  three  smaller  ones  for 
class  use. 

To  mount  the  heart  strip  tie  silk  threads  to  each  end,  one  with  a  loop 
one-half  inch  long  and  the  other  with  a  loop  about  four  inches  long.  Place 
the  short  loop  on  the  hook  of  the  glass-rod  support  provided  for  the  purpose, 
and  the  long  loop  over  the  recording  lever.  Use  a  straw  lever  of  the  power- 
fulcrum-weight  order  mounted  in  a  muscle  lever  holder.  A  total  tension  of 
one  gram  is  best  for  developing  the  contractions  of  the  ventricular  strip. 

The  holder  mentioned  above  is  made  of  a  glass  rod  4  to  5  mm.  diameter 
and  15  cm.  long.  Bend  it  at  a  right  angle  in  the  middle  and  then  draw  out 
and  turn  a  hook  on  one  end,  the  hook  being  turned  back  on  the  rod.  The 
apparatus  set  up  complete  consists  of  a  single  iron  stand  with  three  clamps, 
the  top  one  to  support  the  lever  holder,  the  middle  the  glass  rod,  and  the  bot- 


THE    PREPARATION    OF    THE    VENTRICULAR    MUSCLE. 


67 


FIG.  34. — The  terrapin's  heart,  ventral  view,  showing  how  to  cut  an  apex  strip 
for  experimental  purposes  and  how  to  split  this  apex  into  smaller  pieces. 


FIG.  35. — Apparatus  as  set  up  to  demonstrate  the  contractions  of  the  apex  muscle 
of  terrapin's  ventricle.  The  glass  L-shaped  holder  should  be  set  on  the  stand  high 
enough  to  allow  of  easy  change  of  solution  tubes.  The  figure  shows  the  tube  of  physio- 
logical saline  and  other  details  for  the  better  illustration  of  the  mounting  of  the  heart  strip 


68 


EXPERIMENTAL    PHARMACOLOGY. 


torn  one  a  platform  on  which  rests  the  footed  test-tube  (1x3  inch  specimen 
tube)  to  contain  the  solution  surrounding  the  strip.  The  ventricular  strip 
mounted  in  this  apparatus  with  a  tension  of  one  gram  and  bathed  in  a  solu- 
tion of  0.7  percent  sodium  chloride  will  begin  rhythmic  contractions  in  from 
10  to  40  minutes.  These  contractions  will  continue  about  two  hours, 
growing  constantly  smaller  for  the  entire  time.  The  strip  may  then  be 
revived  by  a  bath  of  Ringer's  solution  or  by  serum,  and  may  again  be  used 
in  the  sodium  chloride  bath. 

TO   TEST   THE   ACTION   OF   DRUGS    ON   THE   FROG'S  OR 
TERRAPIN'S  HEART. 

Two  methods  are  used  in  this  laboratory  for  the  study  of  the  action  of 
drugs  on  the  frog's  heart,  both  permitting  of  permanent  records.     The 


FIG.  36. — Showing  the  method  of  recording  the  action  of  the  frog's  heart  in  place 
n  the  body  cavity.  If  the  perfusion  method  is  used  the  canula  can  be  inserted  with 
greater  ease  if  the  frog  is  reversed  on  the  support. 

most  convenient  method  is  to  pith  the  frog,  open  the  thorax  and  expose 
the  heart,  adjust  the  foot  of  a  delicately  poised  heart  lever  on  the  venticle 
and,  while  the  record  is  being  taken,  irrigate  the  surface  of  the  heart  with  the 


IRRIGATING    AND    PERFUSING    FLASKS.  69 

drug.  Dissolve  the  drug  in  physiological  saline  and  always  take  a  previous, 
normal  record  under  saline  irrigation.  This  method  requires  the  use  of 
relatively  strong  solutions.  The  most  convenient  irrigating  bottles  are  four- 
or  eight-ounce  aspirator  bottles  with  tubed  foot  for  rubber  connector.  These 
are  each  provided  with  a  small-mouthed  canula  attached  by  a  short  rubber 
connection,  and  the  flow  is  regulated  by  a  screw  compress.  Fit  these 
flasks  with  Marriotte  stoppers  and  support  them  on  a  stand  by  a  universal 
buret  clamp  about  the  neck. 

The  second  method,  that  of  perfusion,  is  carried  out  best  as  described 
by  Walden  in  the  American  Journal  of  Physiology,  Volume  III,  page  123. 
Insert  a  canula  into  the  inferior  vena  cava  for  an  inflow  and  one  in  the  aorta 
for  an  outflow,  or  merely  cut  one  aortic  branch  and  let  the  outflow  go  free. 
The  canula  is  connected  with  two  supply  bottles,  one  for  physiological  saline, 
the  other  for  the  drug  in  solution.  The  Marriotte  stoppers  should  be  set 
at  exactly  the  same  pressure  levels.  Connect  the  two  flasks  with  the  inflow 
canula  by  a  T-tube  brought  as  close  as  possible  to  the  heart  in  order  that 
the  solutions  may  be  changed  quickly  with  only  a  short  connecting  tube  to  be 
washed  out.  Very  weak  solutions  of  drugs  are  required  by  this  method  of 
perfusion.  The  frog's  heart  is  quickly  exhausted  in  pure  saline  solutions, 
so,  for  certain  prolonged  experiments  it  is  better  to  use  the  weaker  Ringer's 
solution  for  dissolving  the  drug. 

Record  the  contractions  of  the  ventricle  by  a  thread  from  its  apex  to 
the  ventrical  arm  on  a  balanced  horizontal  lever.  A  flexible  paper  or 
celluloid  writing  point  will  add  to  the  accuracy  and  beauty  of  the  records. 
These  writing  points  should  be  2  to  3  cm.  long  by  0.5  cm.  wide  and  made 
of  light-weight  but  hard  note  paper. 

IRRIGATING  AND  PERFUSING  FLASKS. 

• 

The  quarter-  and  half -pint  aspirator  bottles  manufactured  by  Whitall, 
Tatum  &  Co.,  with  tubed  foot  for  attaching  a  rubber  tube  are  particularly 
adapted  to  both  irrigation  and  perfusion  of  the  heart.  For  use  in  irrigation 
these  bottles  are  clamped  to  a  heavy  based  stand  by  a  universal  buret  clamp 
on  the  neck.  Insert  a  tight-fitting  rubber  stopper  with  a  2  mm.  glass  tube, 
to  give  a  constant  pressure  level.  A  short  heavy  rubber  connector  provided 
with  a  small  screw  compress  and  a  glass  dropper  serves  to  regulate  the  speed 
of  the  outflow.  Such  a  flask  attached  to  an  independent  stand  and  set  at  a 
level  so  that  the  fluid  drops  only  a  few  millimeters  is  an  exceptionally  satis- 
factory method  of  applying  solutions  directly  to  the  surface  of  the  heart. 


70  EXPERIMENTAL    PHARMACOLOGY. 

Two  perfusion  bottles  may  be  connected  together  by  a  T-tube  for  per- 
fusion  work.  In  this  case  the  inflow  canula  is  connected  by  a  very  short 
(6  to  10  cm.)  tube  of  small  caliber  and  is  supported  firmly  by  a  clamp  on 
the  T-tube.  Or  a  Y-canula  can  be  used  and  the  flasks  attached  directly  to 
its  limbs.  In  either  case  it  is  better  to  insert  a  small  T-tube  with  light 
spring  clamp  in  order  to  wash  out  any  drugs  in  the  tubing.  This  canula, 
when  provided  with  an  overflow,  as  shown  by  Gibson  and  Schultz  in  an 
article  now  in  manuscript,  permits  change  from  one  solution  to  another 
without  a  break  in  the  pressure  of  the  fluids.  The  connecting  tubes  for 
the  flasks  should  be  25  to  30  cm.  long  to  permit  adjusting.  Set  screw  or 
spring  compresses  near  the  bottle.  Fill  one  flask  with  the  normal  solution, 
the  other  with  the  drug.  A  very  small  amount  of  the  fluid  can  be  applied 
by  means  of  these  perfusion  flasks. 

TO  TEST  THE  ACTION  OF  DRUGS  ON  THE  BLOOD-PRESSURE, 
RESPIRATION,  ETC.,  OF  A  MAMMAL. 

1.  The  anesthetic.    For  anesthesia  methods  see  page  65. 

2.  The  operations.     Blood-pressure  is  taken  from  one  of  two  arteries, 
the  right  common  carotid,  or  the  femoral  artery.     The  femoral  is  practical 
only  for  the  dog.     To  expose  the  common  carotid  make  a  three-inch  cut 
over  the  trachea  from  the  proximity  of  the  larynx  to  the  manubrium.     Sepa- 
rate the  muscles  down  to  the  trachea,  and  then  along  the  side  of  the  trachea 
till  the  common  carotid  artery  and  vagus  come  into  view.     Use  the  scalpel 
handle  and  tear  rather  than  cut  the  facias  and  muscles  involved.     Avoid 
the  veins  and  the  laryngeal  arteries.     No  blood  need  be  lost  after  the  skin  is 
cut.    Separate  the  fascia  binding  the  artery  and  vagus,  using  care  not  to  injure 
the  latter.     Place  a  bulldog  forceps  on  the  artery  well  toward  the  thorax. 
Ligate  the  cephalic  end.     Lay  and  tie  a  ligature  loosely  about  the  intervening 
stretch  of  artery  for  the  canula.     Grasp  the  artery  at  the  cephalic  ligature 
and  use  the  tip  of  the  scissors  to  make  a  V-shaped  cut  two-thirds  through 
the  artery  wall  and  directed  toward  the  heart.     Insert  the  canula  and  ligate 
it  firmly  with  the  ligature  already  laid. 

The  femoral  artery  is  exposed  by  a  5  cm.  cut  over  the  artery  where 
the  pulse  can  be  felt  near  Poupart's  ligament.  The  artery  is  prepared  and 
the  canula  inserted  as  described  for  the  carotid. 

The  saphenous  vein  or  the  jugular  are  used  for  injecting  drugs. 
Insert  a  small  washout  canula  toward  the  heart  choosing  the  vein  exposed 
by  the  previous  operation.  Keep  the  vein  closed  with  a  bulldog  forceps  in 


ACTION    OF    DRUGS    ON    THE    REFLEXES    OF    A    FROG.  7 1 

order  to  prevent  small  clots  in  the  mouth  of  the  canula,  except  when  in- 
jections are  to  be  made. 

Tracheotomy  should  generally  be  performed  for  all  student  work  on 
the  mammals  used  in  blood-pressure  experiments  in  pharmacology.  Free 
the  trachea  immediately  below  the  thyroid  cartilage  and  insert  a  metal 
canula  made  especially  for  the  dog,  or  insert  one  limb  of  a  glass  T-tube  of 
as  large  size  as  the  trachea  will  take.  Tie  it  firmly  with  small  stout  twine. 

The  apparatus  consists  of  a  continuous  paper  kymograph  (Lud wig's 
weight-driven  pattern  arranged  to  run  the  paper  in  the  right-handed  direction 
is  the  most  satisfactory  instrument) ;  mercury  manometer  for  measuring  the 
blood-pressure;  respiration  tambour  (Marey's  form) ;  signal  pen  to  record 
stimulations,  injections  and  other  events;  time  signal;  stimulating  coil  and 
accessories  complete;  and  a  jacketed  buret  for  transfusing  warm  solutions 
into  the  vein.  The  recording  pens  of  the  manometer,  tambour,  signals,  etc., 
must  all  be  adjusted  to  the  kymograph  in  an  exact  vertical  line.  Fill  the 
lead  tube  of  the  manometer  with  10  percent  magnesium  sulphate  from  a 
pressure  bottle,  take  the  zero  level  of  the  manometer,  set  the  time  signal  to 
write  on  this  level,  connect  with  the  canula,  and  fill  to  a  pressure  of  130  mm. 
mercury.  Connect  the  respiration  tambour  directly  with  the  side  branch  of 
the  tracheal  tube.  Start  the  kymograph,  ink  all  the  pens,  remove  the  arterial 
bulldog  clamp,  and  the  experiment  is  ready  to  begin. 

A  renal  onkometric  record  should  be  taken  with  blood-pressure  in  the 
investigations  on  certain  drugs.  Open  the  abdomen  along  the  entire  median 
line,  cut  the  wall  transversely  for  two  to  three  inches  over  the  left  kidney. 
Strip  the  kidney  of  its  fat  and  looser  coverings  and  enclose  it  in  a  renal 
onkometer.  Adjust  the  overflow  from  the  onkometer  to  the  exact  kidney 
level  and  take  a  record  of  the  variations  with  a  small  sized  Brodie's  bellows 
recorder  adjusted  in  line  with  the  recorders  mentioned  above. 

METHOD  OF  TESTING  THE  ACTION  OF  DRUGS  ON  THE 
REFLEXES  OF  A  FROG. 

Carefully  destroy  the  brain  and  the  medulla,  but  not  the  cord.  Pre- 
vent the  loss  of  blood.  Suspend  the  frog  to  a  horizontal  rod  on  a  stand, 
using  a  card  hanger  or  a  loop  of  string  on  the  upper  jaw.  Stimulate  the  tip 
of  the  toe  with  acid  or  with  platinum  electrodes  and  measure  the  reaction 
time  by  counting  seconds  until  the  foot  is  withdrawn.  The  reaction  time 
may  be  recorded  on  a  kymograph.  Attach  a  horizontal  writing  point  of 
mucilaged  paper  to  the  leg  above  the  foot.  Take  the  speed  of  the  drum  with 


72  EXPERIMENTAL    PHARMACOLOGY. 

one  magnet  beating  seconds,  and  record  the  instant  of  stimulation  with  a 
second  and  independent  magnet  controlled  by  a  contact  key. 
...  ..Take  the  normal  reaction  time  first,  then  give  the  drug  as  an  injection 
in  the  dorsal  lymph  sac  and  allow  about  20  minutes  for  absorption.  Re- 
measure  the  reaction  time  and  repeat  at  intervals  of  10  minutes  to  get  the 
progressive  effects  of  the  drug. 

METHOD  OF  GIVING  AND  TESTING  THE  ACTION  OF  A  DRUG 

ON  THE  FROG'S  GASTROCNEMIUS  MUSCLE. 

diil  £  K"*- 

?,  One  should  always  compare  the  drugged  muscle  with  a  normal  or  un- 
drugged  muscle  from  the  same  frog.  This  may  be  done  in  one  of  two  ways, 
ist.  With  the  circulation  undisturbed.  Pin  the  frog  face  down  on  a  frog- 
board,  isolate  and  attach  the  tendon  Achilles  to  a  muscle  lever,  isolate  and 
stimulate  the  sciatic  at  its  origin  in  the  lumbar  plexus  using  care  not  to  dis- 
turb the  circulation,  or  stimulate  the  muscle  directly.  After  a  normal  record 
is  secured  then  give  the  drug  in  the  usual  way  and  take  a  record  of  the  other 
or  drugged  muscle.  2d.  Lay  a  ligature  about  one  leg  near  the  thigh  tight 
enough  to  stop  its  circulation.  Give  the  drug  by  injection  into  the  dorsal 
lymph  sac,  or  abdominal  cavity,  and  after  absorption  is  complete  and  the 
tissues  have  been  acted  on  by  the  drug  (20  to  30  minutes)  dissect  out  the 
gastrocnemii  and  test.  Always  use  the  undrugged  muscle  first  and  the 
drugged  one  immediately  following.  While  the  normal  muscle  work  is  being 
tested  the  drugged  leg  should  have  its  circulation  stopped  by  ligature.  This 
leaves  the  two  muscles  in  more  nearly  the  same  state  of  nutrition  and 
asphyxiation.  It  is  usually  best  to  stimulate  the  muscle  directly.  There 
are  three  tests  that  can  be  applied:  i,  Irritability,  by  the  minimal  stimu- 
lus method;  2,  Rapidity  of  the  simple  muscle  contraction;  3,  The  amount 
of  work  a  muscle  will  do  with  simple  contractions  at  constantly  repeated 
intervals.  In  this  latter  test  stimulate  once  in  two  seconds,  record  on  a 
drum  with  speed  of  i  mm.  per  second. 

TRANSFUSION  BURET  FOR  MAMMALS. 

Transfusions  of  several  cubic  centimeters  of  liquid  should  be  warmed 
to  body  temperature.  Inclose  a  50  c.c.  buret  in  an  ordinary  Liebig's  con- 
denser jacket  and  mount  vertically  on  a  heavy  base  stand.  Mount  and 
connect  a  6-inch  funnel  with  the  upper  side  tube  of  the  condenser.  Attach 
a  rubber  tube  fitted  with  a  spring  compress  clamp  on  the  lower  side  tube  to 


ARRARATUS    FOR    THE    STUDY    OF    THE    ISOLATED    MAMMALIAN    HEART.       73 

regulate  the  outflow  of  the  warm  water  introduced  by  the  funnel  to  keep  the 
perfusion  liquid  at  the  proper  temperature.  Mount  a  thermometer  inside 
the  condenser  with  its  bulb  near  the  lower  end  of  the  apparatus.  The  buret 
connections  with  the  transfusion  canula  should  be  as  short  as  possible  and 
their  tubes  should  be  provided  with  light  screw-compresses.  Where  only 
i  or  2  c.c.  of  liquid  is  to  be  introduced  it  is  unnecessary  to  warm  it.  In  fact 
a  hypodermic  syringe  is  most  convenient  where  the  volume  of  the  injection 
does  not- exceed  1.5  c.c. 

APPARATUS  FOR  THE  STUDY  OF  THE  ISOLATED  MAMMALIAN 

HEART. 

The  mammalian  heart  isolated  completely  from  the  body  can  be 
maintained  in  constant  activity  for  several  hours.  It  gives  constant  responses 
to  drugs  in  solution  in  the  perfusion  liquid  best  adapted  to  maintain  its 
life,  i.e.,  Locke's  solution  with  a  small  quantity  of  the  animal's  defibrinated 
blood.  Cats  and  rabbits  are  especially  well  adapted  to  this  experiment. 
The  smaller  size  of  the  cat's  or  rabbit's  heart  makes  it  preferable  to  that  of 
a  dog. 

The  points  to  be  secured  in  the  isolated  heart  apparatus  are:  i.  A 
uniform  temperature  of  about  37°  Centigrade.  2.  An  adjustable  pressure 
for  the  perfusion  fluid.  3.  A  device  for  quickly  shifting  from  the  normal 
perfusion  to  the  drugged  perfusion  fluid  without  change  in  temperature, 
pressure  or  any  other  factor  than  the  presence  of  the  drug.  4.  An  accurate 
recording  device. 

The  apparatus  shown  assembled  in  Fig.  37  accomplishes  all  of  the 
above  points.  The  gas  water  heater  connected  as  shown  will  maintain  a 
uniform  temperature  in  the  water  jacket  through  which  the  perfusion  tubes 
run  to  the  heart  canula.  The  overflow  from  the  water  jacket  is  conducted 
into  a  pan  in  which  the  perfusion  fluid  reservoirs  receive  preliminary  warm- 
ing. The  heart  is  attached  to  a  very  short  canula  beneath  the  warming 
jacket  and  the  overflow  of  perfusion  fluid  maintains  a  temperature  of  the 
heart  only  slightly  below  that  of  the  warming  jacket. 

The  pressure  on  the  heart,  i.e.,  on  the  perfusion  fluid,  is  accomplished  by 
connecting  the  perfusion  bottle  with  an  air  or  oxygen  reservoir,  and  this  in 
turn  with  a  water  reservoir  which  can  be  raised  or  lowered.  The  flow  of 
water  from  the  pressure  bottle  into  the  closed  system  produces  the  desired 
pressure  on  the  perfusion  system.  At  the  same  time  the  perfusion  fluids 
are  aerated  by  the  air  (or  oxygen)  as  it  is  forced  into  the  reservoir,  a  result 


74 


EXPERIMENTAL    PHARMACOLOGY. 


accomplished  by  conducting  the  perfusion  bottle  inlet  tubes  to  the  bottom 
of  the  containers. 

A  uniform  pressure  is  secured  on  both  the  normal  and  the  drugged  per- 
fusion fluids  by  the  system  of  tubes  shown.     If  the  clamp  is  removed  from 


FIG.  37. — Illustrating  the  assembly  of  apparatus  for  the  pharmacological  study  of 
the  isolated  heart  of  a  mammal.     The  legends  on  the  apparatus  are  self-explanatory. 


the  outflow  tube  of  the  drugged  perfusion  fluid  at  the  exact  moment  a  second 
clamrj  is  placed  on  the  tube  from  the  normal  fluid  reservoir  (or  vice  versa) , 
the  shift  will  be  accomplished  without  change  of  pressure  on  the  heart.  The 
tubes  run  independently  to  the  canula  which  is  itself  so  short  that  the  time 


LIST   OF    STOCK    SOLUTIONS:.   :  75 

from  the  moment  of  turning  a  perfusion  fluid  On  or J  off  2S  reduced'  to  a  mini- 
mum.    The  canula  is  provided  with  a  side  washout  tube. 

The  Guthrie  cardiograph  shown  is  very  adjustable  in  all  essential  fea- 
tures. It  gives  satisfactory  and  accurate  records,  if  care  is  used  in  inserting 
the  lever  tips  into  the  walls  of  the  heart.  This  apparatus  permits  a  direct 
record  on  the  ordinary  kymograph.  It  also  permits  one  to  surround  the 
heart  with  a  warm  cup  or  jacket  where  greater  constancy  of  temperature  is 
desired,  as  in  research  work. 

LIST  OF  STOCK  SOLUTIONS. 

Make  the  solution  up  in  0.7  percent  sodium  chloride  solution  and  in  Ringer's 
solution.  Special  solutions  must  be  prepared  for  the  mammalian  heart  experiments. 

Aconite  o.i  percent. 

Adrenalin  hydrochloride  o.ooi  percent,  o.oi  percent,  0.05  percent,  o.i  percent. 

Alcohol  95  percent,  2  percent,  5  percent,  10  percent,  20  percent. 

Amyl   nitrite  o.i    percent,   pearls. 

Atropine  o.ooi  percent,  0.002  percent,  o.i  percent,  0.2  percent,  i  percent,  2  per- 
cent, 5  percent,  and  1/120  grain  tablets. 

Barium  chloride  o.oi  percent,  o.i  percent,  0.2  percent,  i  percent. 

Caffeine  o.i  percent,  0.2  percent,  0.5  percent,  i  percent. 

Calcium  chloride  0.03  percent,  0.06  percent,  i  percent. 

Carbolic  acid  0.5  percent,  i  percent,  10  percent. 

Chloral  hydrate  o.i  percent,  i  percent,  2  percent. 

Chloroform  0.05  percent,  o.i  percent,  0.5  percent,  20  percent  in  oil,  pure. 

Cocaine  hydrochlorate  o.oi  percent,  0.2  percent,  0.5  percent,  i  percent,  2  percent. 

Codeine  0.5  percent,  i  percent. 

Curare  0.2  percent,  i  percent. 

Digitalis  0.0005  percent,  o.ooi  percent,  0.002  percent,  o.i  percent,  0.2  percent, 
0.5  percent,  i  percent. 

Ether  i  percent,  2  percent,  4  percent,  6  percent,  8  percent,  pure. 

Ergot  Squibb's  fluid  extract,  10  percent  of  fluid  extract. 

Hyoscyamine  i  percent. 

Locke's  solution. 

Morphine  acetate  i  percent,  2  percent,  10  percent. 

Nicotine  0.02  percent,  o.i  percent,  0.2  percent,  i  percent. 

Nitroglycerine  o.i  percent. 

Physiological  saline  0.7  percent. 

Physostigmine  o.i  percent,  i  percent. 

Pilocarpine  nitrate  o.i  percent,  i  percent,  10  percent. 

Potassium  chloride  0.03  percent,  i  percent,  5  percent. 

Potassium  bromide  5  percent. 

Quinine  hydrochlorate  o.i  percent,  i  percent. 

Ringer's  solution,  weak,  strong. 

Sodium  nitrate  o.oi  percent,  0.02  percent. 

Sodium  sulphate  i  percent. 

Strychnine  nitrate  i  percent,  o.i  percent,  .02  percent. 

Thebaine  0.5  percent,  i  percent. 

Veratrine  0.05  percent,  o.i  percent,  i  percent,  i  percent  of  fluid  extract. 


76  EXPERIMENTAL    PHARMACOLOGY. 

REPORT -FORM  FOR  EXPERIMENTS  ON  FROGS, 

Name 

Date  . 


Animal 

Weight 

Experiment 

i.  Position,  Activity, 
etc. 


I.  NORMAL  REACTIONS. 


2.  Circulation,  Heart, 
Skin,  Mucous  Mem. 


3.  Respiration 


4.  Reflexes 


5,  Eye 


II.  ACTION  OF  THE  DRUG. 

Time Dose How  given Dose  per  kilo. 

Observations  on: 


h.  m  ,s. 


i.  Position  and 
Activities 


2.  Circulation 


3.  Respiration 


4.  Reflexes,  etc. 


5.  Eye 


III.  SUMMARY. 


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OVERDUE. 


BIOLOGY   LIBRARY 


OUT  $2  ISSz 


DEC 
MAR  20  1934 

DEC 


NOV  10 
SEP  lg  1940 

OCT  23  mo 
JAN  2    1962 
\§& 

OCT  28  1969 

OCT  1  5  1969     9 
NOV  19  1970      4 


fillE 


ibject  to  Recall 
Immediately 


LD  21-3m-6. 


BIOLOGY 
UBRARY 

G 
UNIVERSITY  OF  CALIFORNIA  LIBRARY 


